Cd38 modulated chemotaxis

ABSTRACT

The present invention relates to methods for modulating the migratory activity of cells expressing CD38 for the treatment of disorders including, but not limited to, inflammation, ischemia, asthma, autoimmune disease, diabetes, arthritis, allergies, infection with pathogenic organisms, such as parasites, and transplant rejection. Such cells include, for example, neutrophils, lymphocytes, eosinophils, macrophages and dentritic cells. The invention further relates to drug screening assays designed to identify compounds that modulate the ADP-ribosyl cyclase activity of CD38 and the use of such. compounds in the treatment of disorders involving CD38 modulated cell migration. Additionally, the invention relates to the isolation and characterization of a CD38 homologue from the parasitic flatworm,  Schistosoma mansoni.

1. INTRODUCTION

The present invention relates to methods for modulating the migratoryactivity of cells expressing CD38 for the treatment of disordersincluding, but not limited to, inflammation, ischemia, asthma,autoimmune disease, diabetes, arthritis, allergies, infection withpathogenic organisms, such as parasites, and transplant rejection. Suchcells include, for example, neutrophils, lymphocytes, eosinophils,macrophages and dentritic cells. The invention further relates to drugscreening assays designed to identify compounds that modulate theADP-ribosyl cyclase activity, NAD glycohydrolase activity, andtransglycosidation activity of CD38 and the use of such compounds in thetreatment of disorders involving CD38 modulated cell migration. Thepresent invention relates to the isolation and characterization of aCD38 homologue from the parasitic flatworm, Schistosoma mansoni. Theidentification of such a homologue, referred to herein as SM38 or SARC,provides compositions and assays designed to screen for related enzymesin pathogenic organisms as well as compositions and assays to screen forcompounds that modulate the activity and/or expression of SM38. Suchcompounds can be used to treat pathogenic disorders resulting frominfection with such parasites. The invention is based on the discoveriesthat CD38 ADP-ribosyl cyclase activity is required for chemotaxis andthat S. mansoni expresses a CD38 homologue that can regulate calciumresponses in the parasite.

2. BACKGROUND OF INVENTION

Hematopoietically-derived cells, including cells such as neutrophils,monocytes, dendritic cells, eosinophils and lymphocytes, are importantcellular mediators of the inflammatory response and respond to solubleinflammatory mediators by migration to the site of tissue injury orinfection where the newly arrived cells perform their effectorfunctions.

Neutrophils which represent 40-50% of the circulating leukocytepopulation are particularly important to both immunity and inflammation.Neutrophils are normally quiescent cells but upon stimulation canmediate a variety of different inflammatory activities. A large numberof different agents are capable of activating neutrophils and thisactivation is normally mediated by binding of the activating agent tospecific receptors expressed on the surface of neutrophils. Onceactivated, the neutrophils are capable of binding to endothelial cellsand migrating to the site of tissue damage, a pathogen or a foreignmaterial. Similarly, eosinophils are also potent inflammatory effectorcells, although these cells are most often associated with allergicdiseases such as asthma. Like neutrophils, eosinophils have a potentarmory of proinflammatory molecules that can initiate and maintaininflammatory responses.

Once at the inflammatory site, recruited cells such as eosinophils andneutrophils induce further inflammation by releasing inflammatoryproducts and recruiting other hematopoietically-derived cells to thesite. In some cases, the inflammatory response mediated by thespecifically recruited hematopoietically-derived cells protects the hostfrom morbidity or mortality by eliminating the infectious agent. Inother cases (i.e., autoimmunity, ischemia/reperfusion, transplantation,allergy), the inflammatory response further damages the tissue resultingin pathology. Thus, agents which alter inflammation or recruitment ofcells may be useful in controlling pathology.

Although CD38 expression was at first believed to be restricted to cellsof the B cell lineage, subsequent experiments by a number of groups havedemonstrated that CD38 is widely expressed on both hematopoietic andnon-hematopoietically-derived cells. Homologues of CD38 have also beenfound to be expressed in mammalian stromal cells (Bst-1) and in cellsisolated from the invertebrate Aplysia californica (ADP-ribosyl cyclaseenzyme) (Prasad G S, 1996, Nature Structural Bio 13:957-964)

More recently, CD38 was shown to be a multifunctional ecto-enzyme withNAD+glycohydrolase activity, transglycosidation activity and ADP-ribosylcyclase activity, enabling it to produce nicotinamide, ADPribose (ADPR),cyclic-ADPR (cADPR) and nicotinic acid adenine dinucleotide phosphate(NAADP) from its substrates NAD+ and NADP+ (Howard et al., 1993 Science252:1056-1059; Lee et al., 1999 Biol. Chem. 380; 785-793). Cyclic ADPRmediates intracellular calcium release through ryanodine receptor gatedstores (Galione et al., 1991 Science 253:1143-1146; Lee, 1993 J. Biol.Chem. 268:293-299; Meszaros et al., 1993 Nature 354:76-78), while ADPRinduces Ca²⁺ influx in mammalian cells by activating the plasma membraneion channel, TRPM2 (Perraud et al. 2001 Nature: 411:595-599; Sano et al.2001 Science 293:1327-1330; Hara et al. 2002 Mol. Cell. 9:163-173). Inaddition, NADP⁺, which is also utilized as a substrate by cyclases, canbe transformed into nicotinic acid adenine dinucleotide (NAADP⁺) in abase-exchange reaction in the presence of nicotinic acid (Aarhus et al.1995. J. Biol. Chem. 270:30327-30333). NAADP⁺ is a very powerfulCa²⁺-mobilizing metabolite that mediates Ca²⁺ release from intracellularstores that are gated independently of both IP₃R and RyRs (Lee et al.,1995 J. Biol. Chem. 270:2152-2157). Thus, cyclases have the ability toproduce at least three different second messengers that mobilizemultiple independent sources of calcium, suggesting that thesemetabolites may be global regulators of calcium responses (Lee et al.,1999 Biol. Chem. 380; 785-793). All three of these second messengers arealso produced by SM38.

Both cADPR and NAADP are known to induce calcium release from calciumstores that are distinct from those controlled by IP3 receptors(Clapper, D L et al., 1987, J. Biological Chem. 262:9561-9568). Instead,cADPR is believed to regulate calcium release from ryanodine receptorregulated stores, as agonists of ryanodine receptors sensitize cADPRmediated calcium release and antagonists of ryanodine receptors blockcADPR dependent calcium release (Galione A et al., 1991, Science253:143-146). Thus, it has been proposed that cADPR is likely toregulate calcium responses in tissues such as muscle and pancreas whereryanodine receptors are expressed. Interestingly, it was recently shownthat the muscle fibers of the parasitic flatworm, S. mansoni, expressryanodine receptors and that agonists of ryanodine receptors such ascaffeine can induce intracellular calcium release and muscle contractionin the parasite (Day et al., 2000 Parasitol 120:417-422; Silva et al.,1998, Biochem. Pharmaco. 156:997-1003). In mammalian smooth musclecells, the calcium release in response to acetylcholine can be blockednot only with ryanodine receptor antagonists, but also with specificantagonists of cADPR such as 8-NH2-cADPR or 8-Br-cADPR (Guse, A H, 1999,Cell. Signal. 11:309-316).

These findings, as well as others, indicate that ryanodine receptoragonists/antagonists including cADPR can regulate calcium responses incells isolated from species as diverse as helminths to mammals, however,it is unclear whether ADP-ribosyl cyclase enzymes such as CD38 or SM38are required for the production of cADPR in vivo. Additionally, therehas been no direct evidence to link CD38 enzyme activity with downstreamresponses such as calcium release, proliferation, apoptosis, migrationor other effector functions. Thus, despite the high level expression ofCD38 on many cell types, no clear defining role for CD38 enzyme activityin immune responses has been established.

3. SUMMARY OF THE INVENTION

The present invention relates to methods for modulating the migratoryactivity of cells expressing CD38 involving the administration ofagonists or antagonists of CD38 enzyme activity, and the cADPR mediatedsignal transduction pathway, including small molecules, large molecules,and antibodies. The invention also provides for compounds and nucleotidesequences that can be used to modulate CD38 gene expression.

The present invention further relates to the isolation andcharacterization of a CD38 homologue from the parasitic flatwormShistosoma mansoni, herein referred to as SM38. The identification ofsuch a homologue provides compositions and assays designed to screen forrelated enzymes in pathogenic micro-organisms (such as helminths) aswell as compositions and assays to screen for compounds that modulatethe activity of SM38. Such compounds can be used to treat pathogenicdisorders resulting from infection with such pathogenic micro-organisms.

The invention relates to assays designed to screen for compounds thatmodulate the enzymatic activity of CD38 and/or SM38 (CD38/SM38), i.e.,compounds that act as agonists and antagonists of CD38 enzyme activity.When screening for such compounds for treatment of helminth infection,it is preferred that the compound selectively inhibit SM38 and not CD38.In addition, the screens of the invention may be used to identifysubstrates of CD38/SM38 that are converted into antagonists or agonistsof signal transduction pathways involving cADPR. The screens of theinvention also maybe used to directly identify agonists and antagonistsof signal transduction pathways involving cADPR.

The invention also relates to assays designed to screen for compoundsthat modulate CD38/SM38 gene expression. For example, cell-based assayscan be used to screen for compounds that modulate CD38/SM38transcription such as compounds that modulate expression, production oractivity of transcription factors involved in CD38/SM38 gene expression;antisense and ribozyme polynucleotides that modulate translation ofCD38/SM38 mRNAs and polynucleotides that form triple helical structureswith the CD38/SM38 regulatory regions and inhibit transcription of theCD38/SM38 gene.

Identified compounds may be used in the treatment of disorders where themigratory activity of CD38-expressing cells, such ashematopoietically-derived cells, contributes to the development of suchdisorders. Such disorders include, but are not limited to inflammation,ischemia, asthma, autoimmune disease, diabetes, arthritis, allergies ortransplant rejection where inhibition of migratory activity using, forexample, CD38 antagonists would be desired. In contrast, in subjectsinfected with pathogenic microorganisms or immunosuppressed subjects itmay be, desirable to induce the migratory activity ofhematopoietically-derived cells using, for example, agonists of CD38.Additionally, identified compounds may be used to treat pathogenicdisorders resulting from infection with pathogenic micro-organismsexpressing SM38 or structurally related homologous proteins.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Normal Cellular Response to Chemoattractant Signaling. (1)Chemoattractant binds to receptor and initiates signaling. (2) CD38hydrolyzes NAD and produces cADPR, which facilitates Ca2+ release frominternal stores. (3) Ca2+ is released from cADPR-controlled internalstores which activates external Ca2+ channel. (4) Extracellular Ca2+flows into the cell and allows migration.

FIG. 2. Inhibitors of cADPR Production by CD38 Prevent Capacitative Ca2+Entry and Chemoattractant Induced Migration (Screens will identify suchcompounds). (1) Chemoattractant binds to receptor and initiatessignaling. (2) Inhibitor of CD38 prevents either hydrolysis of NAD(enzyme is inactive and no products are made) or specifically inhibitsproduction of cADPR (blocks ADP-ribosyl cyclase activity, but enzyme maynot be inactive). (3) Lack of cADPR results in no cADPR-mediated Ca2+release from internal stores. (4) No capacitative Ca2+ influx and nomigration.

FIG. 3. Proteins that Regulate CD38 Enzyme Activity (Screens willidentify compounds that activate or inactivate these proteins). (1)Chemoattractant binds to receptor and initiates signaling. (2) ProteinX. modifies CD38 and inactivates CD38 enzyme activities. (3) Lack ofcADPR results in no cADPR-mediated Ca2+ release from internal stores.(4) No capacitative Ca2+ influx and no migration.

FIG. 4. Proteins that Regulate CD38 Expression (Screens will identifycompounds that activate or inactivate these proteins). (1)Chemoattractant binds to receptor and initiates signaling. (2) Protein Xrepresses CD38 gene transcription. (3) Lack of CD38 results in absenceof cADPR which results in no cADPR-mediated Ca2+ release from internalstores. (4) No capacitative Ca2+ influx and no migration.

FIG. 5. Alternate Substrates for CD38 may generate inhibitors of cADPRand prevent capacitative Ca2+ release (Screens will identify suchcompounds). (1) Chemoattractant binds to receptor and initiatessignaling. (2) CD38 hydrolyzes modified substrate (8-BrNAD, for example)and produces modified product (8-Br-cADPR, for example) (3) Modifiedproduct competitively or non competitively inhibits cADPR induced Ca2+release from internal stores. (4) No capacitative Ca2+ influx and nomigration.

FIG. 6. Inhibitors of cADPR binding block capacitative Ca2+ influx. (1)Chemoattractant binds to receptor and initiates signaling (Screens willidentify such compounds). (2) CD38 hydrolyzes NAD and produces cADPR.(3) Inhibitor of cADPR (8-Br cADPR) competitively or non-competitivelyblocks cADPR induced Ca2+ release from internal stores. (4) Nocapacitative Ca2+ influx and no migration.

FIG. 7. CD38KO mice are more susceptible to S. pneumoniae infection. (a)C57BL/6 WT (open circles) and CD38KO (filled circles) mice were infectedintra-tracheally with two doses of S. pneumoniae. The survival ofinfected animals was monitored over the next 4 days. (b) WT mice thathad been irradiated and reconstituted with WT bone marrow (open squares)or CD38KO bone marrow (filled squares) were infected with two doses ofS. pneumoniae and monitored for four days. The data are representativeof at least 5 independent experiments. n=10 mice/group. (c) WT or Rag-2KO (open bars) and CD38KO or CD38-Rag-2 double KO (filled bars) micewere infected intra-tracheally with S. pneumoniae and bacterial titersin lung and peripheral blood were determined at 12 hours post-infection.The data are representative of 3 independent experiments. N=10mice/group. *P<0.001; Student's t test.

FIG. 8. CD38KO neutrophils are not recruited to the infection site andare unable to chemotax toward bacterially-derived chemoattractants. WTand CD38KO mice were infected intra-tracheally with S. pneumoniae, andthe cellular infiltrate in the airways was collected and counted (panela, WT=open bars and CD38KO=closed bars) at multiple timepointspost-infection. (b) The identity and frequency of the infiltrating cellsin the lungs of infected WT and CD38KO mice was determined bymicroscopic examination (400× magnification) and counting of Diff-Quickstained cytocentrifuge preparations. (c) Differential cell counts in thelung lavage of WT (open bars) and CD38KO (closed bars) mice arepresented as the mean number of cells×10⁶±(SE). Similar results wereobtained in 5 independent experiments. n=5 mice/group/timepoint. *P<0.01**P=0.01; Student's t Test. (d) Purified bone marrow neutrophils from WT(open bars) and CD38KO mice (filled bars) were tested for their abilityto migrate in response to medium, fMLP or IL-8 in a conventionaltranswell checkerboard chemokinesis/chemotaxis assay. The number ofcells migrating to the bottom chamber of the transwell in the absence ofany stimulation was not significantly different between CD38KO and WTneutrophils and ranged from 1500-2300 cells (not shown). The number ofneutrophils migrating in response to equivalent concentrations ofstimuli in both chambers (chemokinesis) and the number of neutrophilsmigrating in response to a chemotactic gradient (chemotaxis) is shown.The values shown are the mean±S.E. of four different experiments.*P<0.001; Student's t Test.

FIG. 9. CD38 expressing neutrophils produce cADPR and releaseintracellular calcium in response to cADPR and ryanodine. (a) Bonemarrow, peripheral blood and peritoneal cavity cells were isolated fromWT and CD38KO mice or WT and CD38KO mice that received anintraperitoneal injection of thioglycollate 12 hrs previously. CD38expression on the Mac-1^(hi)GR 1^(hi) neutrophils was analyzed by flowcytometry. Expression of CD38 on WT neutrophils (solid line histogram)and CD38KO neutrophils (dotted line histogram) is shown. (b) CD15+ humanperipheral blood neutrophils were assessed for CD38 expression bystaining with anti-CD38 mAb (filled histogram) or an isotype control Ab(dotted line). (c) Cyclase activity was measured in WT and CD38KO bonemarrow neutrophils. WT or CD38KO neutrophils were incubated alone(WT=circles and CD38KO=squares) or in the presence of NOD (WT=trianglesand CD38KO=diamonds) for 10 minutes. The accumulation of the product,cGDPR, was measured fluorometrically. (d) RyR3 mRNA expression levelswere determined by RT-PCR. cDNA was isolated from purified WT bonemarrow neutrophils (PMN) or brain tissue. The amount of input cDNA isindicated. (e-g) Intracellular free calcium levels were measured by FACSin Fluo-3/Fura Red loaded bone marrow neutrophils. (e) Neutrophils werepermeabilized with digitonin and then stimulated with ryanodine in thepresence (orange line) or absence (blue line) of ruthenium red. (f)Neutrophils were permeabilized in digitonin and then stimulated withcADPR (blue line), heat inactivated cADPR (green line) or8-Br-cADPR+cADPR (red line), (g) Neutrophils were stimulated withthapsigargin (blue line) or thapsigargin+8-Br-cADPR (red line). All datain panels' e-g are representative of at least three independentexperiments.

FIG. 10. CD38 catalyzed cADPR regulates intracellular calcium release,extracellular calcium influx and chemotaxis in neutrophils. (a-c)Intracellular free calcium levels were measured by FACS in Fluo-3/FuraRed loaded bone marrow neutrophils. (a) CD38KO (red line) and WT (blueline) neutrophils were stimulated with fMLP or IL-8 in calcium-freebuffer. (b) CD38KO (red line) and WT (blue line) neutrophils werestimulated with fMLP or IL-8 in calcium-containing buffer. (c) CD38KO(red line) and WT (blue line) neutrophils were preincubated incalcium-containing medium±8-Br-cADPR and then stimulated with fMLP orIL-8. All data in panels a-c are representative of at least fiveindependent experiments. (d) WT neutrophils were pre-incubated withmedium, EGTA or 8-Br-cADPR and then placed in the top chamber of atranswell that contained fMLP or IL-8 in the bottom chamber. The cellsthat migrated to the bottom chamber in response to the chemotacticgradient were collected and enumerated by flow cytometry. Values shownare mean±S.E. from three separate experiments with threewells/experimental condition. **P=0.008; Mann Whitney Rank Sum Test.

FIG. 11. An NAD+analogue regulates calcium influx and chemotaxis infMLP-activated neutrophils. (a) Dye-loaded purified bone marrowneutrophils from WT mice were preincubated in medium (blue line) orincreasing concentrations of N(8-Br-A)D+(red line) and then stimulatedwith fMLP. Changes in intracellular calcium levels were measured by flowcytometry. The data are representative of three independent experiments.(b-c) WT (left panel) and CD38KO (right panel) neutrophils werepreincubated with medium (filled bars) or N(8-Br-A)D+(open bars) andthen placed in the top chamber of a transwell which contained fMLP(panel b) or IL-8 (panel c) in the bottom chamber. The cells thatmigrated to the bottom chamber in response to the chemotactic gradientwere collected and enumerated by flow cytometry. Values shown are mean tS.E. from three separate experiments with three wells/experimentalcondition. *P<0.001 Student's t Test.

FIG. 12 A-B. The recruitment of neutrophils and eosinophils to the lungsin a model of allergic asthma is impaired in CD38 KO mice. Naive CD4 Tcells from WT C57BL/6 mice or OVA-primed CD4 T cells from WT C57BL/6mice were transferred to either WT C57BL/6 mice or to CD38KO-057BL/6mice as indicated. Recipient mice were subsequently challenged on 7consecutive days by intratracheal instillation of 10 μg OVA in PBS.Neutrophils (A) and eosinophils (B) in the lung lavage on the eighth dayafter initial challenge were enumerated by microscopic examination(400×) of Diff—Quick stained cytocentrifuge preparations

FIG. 13. Identification and isolation of SARC cDNA. FIG. 13A. A fulllength cDNA encoding was cloned from a S. mansoni cDNA library using acloned S. mansoni EST (Accession # AW017229) that was identified in ablast search using the consensus ADP-ribosyl cyclase family sequence.The putative initiation methionine(s) are indicated in green and thestop site is indicated in red. The primers used to clone the originallyidentified EST are shown in blue and the amino acids within the EST thatare absolutely conserved within the cyclase family are indicated inyellow. FIG. 13B. Comparison of the amino acid sequence of S. mansoniSM38 with representative members of the ADP-ribosyl cyclase family. Theabsolutely conserved amino acids are indicated with * and conservativereplacement amino acids are indicated with (:). The % identity and %similarity are indicated. The 10 conserved cysteine residues requiredfor intradisulfide bonds and protein folding are indicated in red. Thehighly conserved “signature domain” within the active site of cyclasefamily members is shown in yellow, a critical substrate bindingtryptophan residue is shown in green and the key catalytic glutamateresidue is indicated in blue.

FIG. 14. SM38 is homologous to Aplysia ADP ribosyl cyclase and humanCD38 cyclase. The protein sequence of SM38 was aligned with the proteinsequences for Aplysia ADP-ribosyl cyclase (part a) and human ADP-ribosylcyclase CD38 (part b). A high degree of homology (boxed residues) wasobserved with 21% identity between the Aplysia protein and SM38 and 23%identity between human CD38 and SM38. The conserved 10 cysteine residuespresent in all members of the cyclase protein family are also present inSM38 (shaded boxes). The two additional cysteines found in CD38(underlined), but not in Aplysia, are also lacking in SM38. However, theSM38 protein contains two additional cysteine residues that are uniqueand are not found in either CD38 or Aplysia cyclase (underline). Mostimportantly, the active site catalytic residues identified for CD38 andAplysia enzyme (starred residues) are also present in SM38.

FIG. 15. Amino acid sequence comparison between SM38 cloned from S.mansoni and a homologous protein identified from S. japonicum (Accession# AY222890). The conserved amino acids are indicated with * and theconservative replacement amino acids are indicated with a (:). The %identity and % similarity are indicated. The signal sequence, identifiedby the SignalP prediction program (Bendtsen, J. D. et al., 2004 J MolBiol; 340:783-795), is indicated in green and the potential GPI anchorsequence (Eisenhaber, et al., 1998, Protein Eng., 11:1155-1161) isindicated in magenta with the probable ω site for GPI addition shown inyellow. The 10 cysteine residues that are conserved among all cyclasefamily members are indicated in red and the two additional cysteineresidues found only in Schistoma proteins are shown in orange. Theinvariant catalytic glutamate (E202) residue is shown in blue and thefour potential N-linked glycosylation sites are in bold and italics.

FIG. 16. Immunoreactivity of SM38 polyclonal antibodies with the nativeprotein in schistosome extracts. Affinity purified SM38-polyclonal andnormal mouse IgG were used to probe schistosome extracts separated onto12% agarose gel. Panel I shows a Coomassie blue stainedSDS-polyacrylamide gel (molecular weight range between and 33-45 kDa) ofS. mansoni whole adult worm (lane 1), carcass (lane 2) and NP-40 (lane3) extracts. Panel II shows reactivity of normal mouse IgG withschistosome extracts. No specific reactivity was found. Panel III showsthat anti-SM38 mouse IgG detected a specific protein of an apparentmolecular weight of about 38 kDa in all extracts tested.

FIG. 17. Reverse translation of SM38. The 303 amino acid coding regionof SM38 was reverse-translated to identify a degenerate DNA sequencethat would encode the SM38 protein.

FIG. 18. SM38 is a highly conserved protein expressed by two Schistosomaspecies. FIG. 18A. Phylogenetic comparison of cyclase family members.The amino acid sequences of the 11 previously identified members of thecyclase family (see Experimental Procedures for Accession #s) werecompared to the two novel SM38 sequences and assembled into aphylogenetic tree. FIG. 18B. Proposed three dimensional structure ofSM38. A homology model was constructed based on the crystallographiccoordinates of both Aplysia ADP-ribosyl cyclase (PDB entry 1lbe) andhuman CD157 (PDB entry 1isf) using Modeller (50) and energy minimizationusing AMBER5. A ribbon representation of monomeric SM38 (green) issuperimposed over that of human CD157 (red). The nicotinamide bound toCD157 (PDB entry 1ism) is shown as space filling model. Carbon atoms arecolored in white, oxygen atoms in red, nitrogen atoms in dark blue andhydrogen atoms in cyan. FIG. 18C, Connoly surface of the putative activesite of SM38. The surfaces of four important amino acids are highlightedusing the color code defined in C. The amino acid residues shown includethe putative catalytic Glu²⁰², the Glu¹²⁴ that regulates the ADP-ribosylcyclase activity of CD38 and the substrate-binding Trp¹⁶⁵ and His¹⁰³.The rendering was performed using SYBYL (Tripos Inc.).

FIG. 19. S. mansoni SM38 is a GPI-anchored NADase when heterologouslyexpressed in mammalian cells. FIG. 19A. Native SM38 is cell-associated.COS-7 cells were transiently transfected with the full length SM38(SM38-opt). After three days, the culture media (circles) and cells werecollected separately. The cells were lysed and the detergent solubleproteins were collected (squares). Aliquots of the cell lysate and theconditioned tissue culture media were incubated with ε-NAD⁺ andconversion of ε-NAD⁺ to fluorescent ε-ADPR was measured over time in amicroplate fluorimeter. Data is represented in relative fluorescentunits (RFU) vs time. No enzyme activity was observed in non-transfectedCOS-7 cells or COS-7 cells transfected with the empty vector (data notshown). FIG. 19B. SM38 is expressed as a ˜48 kD protein in COS-7 cells.The native signal sequence of S. mansoni SM38 was replaced with themammalian CD8 signal sequence and a FLAG tag (CD8L/FLAG-SM38). Celllysates of COS-7 cells transiently transfected with the CD8L/FLAG-SM38construct were analyzed by western blot using an anti-FLAG antibody toidentify SM38. FIGS. 19C-D. SM38 is expressed as a plasmamembrane-associated protein in transfected COS-7 cells. COS-7 cells weretransiently transfected on slides with CD8L/FLAG-SM38 (panel C) or theempty expression vector (panel D). The transfected cells were fixed andstained with a biotinylated anti-FLAG antibody followed by fluorochromecoupled strep-avidin (red) and a nuclear counterstain (DAPI, blue).Cells were analyzed by fluorescent microscopy. FIG. 19E. SM38 isexpressed as a GPI-anchored protein in COS-7 cells. COS-7 cells weretransiently transfected with either CD8L/FLAG-SM38 (circles andtriangles) or the control expression vector (squares and diamonds).Three days later the transfected cells were washed and then cultured infresh media (squares and circles) or fresh media containing PI-PLC(diamonds and triangles). Two hours later the media was collected andincubated in the presence of ε-NAD⁺. Conversion of ε-NAD⁺ to fluorescentε-ADPR was measured in a microplate fluorimeter and is reported as RFUvs time.

FIG. 20. Recombinant soluble SM38 catalyzes NAD glycohydrolase, cyclaseand transglycosidation reactions. FIG. 20A. Recombinant soluble SM38 issecreted. COS-7 cells were transiently transfected withCD8L/FLAG-SM38ΔGPI, a construct lacking the GPI anchor sequence.Transfected COS-7 cells were lysed, detergent soluble proteins werecollected and aliquots of the cell lysate (squares) and culture media(circles) were collected. Aliquots were then incubated with ε-NAD⁺ andconversion and accumulation of fluorescent ε-ADPR was measured in amicroplate fluorimeter. The remaining culture media was purified over ananti-FLAG column and aliquots were again tested for NADase activityusing ε-NAD* as the substrate (inset). Data is reported in RFU vs time.FIG. 20B. Recombinant soluble SM38 is glycosylated in mammalian cells.Recombinant soluble SM38 was purified from COS-7 cells transientlytransfected with CD8L/FLAG-SM38ΔGPI. Affinity purified SM38 wasincubated in the presence or absence of Endoglycosidase-F1 (Endo-F) andthe proteins were separated by SDS-PAGE and analyzed by silver staining(left panel) or western blotting with anti-FLAG antibody (right panel).The molecular weight of soluble recombinant SM38 is indicated. FIG. 20C.Recombinant soluble SM38 is expressed in Pichia pastoris. Pichia weretransformed with an expression vector containing the SM38 ecto-domain. Astable clone was selected and SM38 production and secretion was inducedwith methanol. Secreted SM38 was purified from the media bychromatography and analyzed by SDS-PAGE and Coomassie staining. Themolecular weights of the purified proteins are 45.2 (*) and 43.6 (**)kDa. FIG. 20D. Soluble recombinant SM38 catalyzes the transformation ofNAD⁺ to ADPR. Recombinant soluble SM38 was purified from Pichia and thenincubated with radio-labeled NAD⁺. The accumulation of radio-labeledADPR and cADPR was measured by HPLC. FIGS. 20E-F. Soluble recombinantSM38 catalyzes the transformation of NGD⁺ to cyclic GDP-ribose. Purifiedrecombinant soluble SM38 was incubated with increasing quantities ofNGD⁺ and the accumulation of cyclic GDPR and GDPR was detected by HPLCand UV detection (260 nm) at various time points. 20G. SM38 catalyzesthe transglycosidation of NADP⁺ to NAADP⁺. NADP⁺ (1 mM) was incubatedwith recombinant purified SM38 at 37° C. in the presence of 20 mMnicotinic acid (NA). Aliquots were analyzed by HPLC. The compounds weredetected by UV absorbance at 260 nm. ADPR(P), adenosine diphosphoribose2′-phosphate.

FIG. 21. SM38 expression is developmentally regulated in S. mansoni.FIG. 21A. cDNA prepared from RNA isolated from multiple developmentalstages of S. mansoni was used as the template for RT-PCR reactions usingSM38-specific primers. Schistosome specific α-tubulin primers were usedto amplify a constitutively transcribed internal control gene. Testedstages are numbered from 1 to 12 and they represent the following:uninfected B. glabrata, 30-day infected B. glabrata, S. mansoni eggs, S.mansoni cercariae, S. mansoni 15-day schistosomules, 21-dayschistosomules, 28-day worms, 35-day worms, adult (>42-day old) wormpairs, adult female worms, adult male worms and no reverse transcriptase(—RT) control, respectively. FIG. 21B. Bar-graph representation of theaverage expression level of SM38 exhibited by each tested developmentalstage of the parasite life cycle as percentage of levels of α-tubulininternal control. Data shown are averages of values quantified fromthree independent PCR amplifications. Background (—RT control) wassubtracted from each of the analyzed samples.

FIG. 22. Adult S. mansoni worms express a GPI-anchored NADase on theouter membrane. FIGS. 22A-C. Adult S. mansoni worms express aGPI-anchored NAD⁺ glycohydrolase and NGD⁺ cyclase. Membrane microsomeswere prepared from 2 g of frozen adult S. mansoni worms. The microsomeswere resuspended in buffer and incubated with ¹⁴C-labeled NAD⁺ (A) orunlabeled NGD⁺ (B). Product formation was measured by HPLC as describedin FIG. 20. In panel C, the membrane microsomes were incubated in thepresence or absence of PI-PLC for two h. The supernatant and membranefractions were collected separately and then incubated with ¹⁴C-labeledNAD⁺. ADPR production was measured by HPLC and results are presented as% activity compared to the non-treated membrane fraction. The specificactivity of non-treated membrane microsomes was 36 nmol/min/mg protein.FIG. 22D. Adult S. mansoni worms express an outer membrane NAD⁺glycohydrolase. Ten live adult S. mansoni worms were placed in singlewells of a 96 well plate and were incubated in media (circles) or mediacontaining ε-NAD* (squares) and conversion of ε-NAD⁺ to fluorescentε-ADPR was measured in a microplate fluorimeter and is reported in RFUvs time. FIG. 22E. Adult S. mansoni worms express a GPI-anchored outertegument NADase. Ten live adult S. mansoni worms were placed in singlewells of a 96 well plate and were then incubated in the presence(diamonds and triangles) or absence (squares and circles) of PI-PLC for2 h. The buffer from each of the wells was removed and then incubated inthe presence (circles and triangles) or absence (squares and diamonds)of ε-NAD⁺. Production of fluorescent c-ADPR was measured in a microplatefluorimeter and is reported as RFU over time.

FIG. 23. Antiserum raised against the SM38 cDNA immunoprecipitatesenzymatically active SM38 from adult S. mansoni worms. FIG. 23A.Antibodies raised in response to SM38 cDNA immunization recognizerecombinant soluble SM38. Control serum (IgG) and antiserum collectedfrom mice vaccinated with the CD8L/FLAG-SM38ΔGPI construct (anti-SM38)were used to probe western blots containing recombinant soluble SM38 orirrelevant protein (ovalbumin, OVA). FIGS. 23B-D. Antiserum raised inresponse to immunization with SM38 cDNAs specifically recognize plasmamembrane-associated SM38. COS-7 cells were transiently transfected onslides with CD8L/FLAG-SM38 (C-D) or the empty expression vector (B).Three days later the cells were stained with anti-SM38 antiserum (B, D)or normal mouse serum (C) followed by fluorochrome coupled anti-mouseIgG (red) and a nuclear counterstain (DAPI, blue), FIG. 23E. Anti-SM38antibodies immunoprecipitate functional SM38 protein from transfectedCOS-7 cells. COS-7 cells were transiently transfected withCD8L/FLAG-SM38 (squares and circles) or the empty expression vector(diamonds and triangles). Three days later the cells were lysed and thelysates were incubated with either normal mouse IgG protein G beads(diamonds and squares) or with anti-SM38 protein G beads (triangles andcircles). The immunoprecipitated protein/bead complexes were incubatedin the presence of ε-NAD⁺ and the accumulation of fluorescent ε-ADPR wasmeasured using a microplate fluorimeter. Data is reported in RFU vstime. 23F. Antibodies raised in response to SM38 cDNA immunizationimmunoprecipitate enzymatically active SM38 from adult S. mansonilysates. Adult S. mansoni worms were lysed in detergent and the lysateswere incubated with either normal mouse IgG protein G beads (triangles)or with anti-SM38 protein G beads (diamonds). The immunoprecipitatedprotein/bead complexes were incubated in the presence of s-NAD⁺ and theaccumulation of fluorescent ε-ADPR was measured using a microplatefluorimeter. Data is reported as RFUs vs time. FIG. 23G. Antibodiesraised in response to SM38 cDNA immunization recognize a GPI-anchoredprotein expressed by adult S. mansoni worms. Live adult worms wereincubated in HBSS in the presence of PI-PLC for 2 h. The supernatant wascollected, concentrated and then electrophoresed on a SDS-PAGE gel. 15%of the total protein was analyzed by silver staining and western blotusing anti-SM38 anti-serum. Control lanes include PI-PLC alone andincreasing concentrations of recombinant soluble SM38.

FIG. 24. Immunohistochemical localization of SM38 in adult S. mansoni.Adult 87 schistosome cryosections (A-F) and live or acetone-fixedwhole-mount adult worms (G-1) were prepared. Panels A and D representphase contrast fields. Samples visualized using a green fluorescencefilter (Panels B and E) indicate auto-fluorescent structures includingfemale worm vitellaria (Panel B). To localize SM38, sections and wholemounts were stained with the IgG fraction purified from the anti-SM38antiserum (Panels F, H, I) or with normal mouse IgG (Panels C, G)followed by staining with anti-mouse IgG and Alexa Fluor 647 conjugatedstreptavidin. SM38 was visualized using a far-red fluorescence filter.Slides stained with normal mouse IgG did not show any specificreactivity (Panel C, G), while slides stained with anti-SM38 antibodyshowed specific staining in the tegument layer of male worm gynecophoriccanal (arrow, Panel F). Acetone-fixed whole adult worms (male wormshown) stained with anti-SM38 mouse IgG showed strong overallfluorescence (Panel H), while in live whole worms (female shown, PanelI) fluorescence was more localized to oral sucker (Os), ventral sucker(Vs) and tubercles (Tu). V stands for vitellaria, G is gut, T istegument and GC is male gynecophoric canal. Sections and whole wormswere photographed at 200× and 100× magnifications, respectively.

FIGS. 25-26. CD38 expression on allergen-specific T cells (or autoimmuneT cells) is required for either their maturation into differentiatedeffector cells or for their migration to sites of inflammation.OVA-specific T cells which are CD38 deficient are reduced in number inboth the lymph node and at the site of inflammation in the lungs of micesensitized with 10 μg NP-OVA (n=7 mice/group) or PBS administered (n=3mice/group). Similarly, the allergen-induced inflammatory response issuppressed in the lungs of mice receiving CD38 deficient T cells. Thenumber of donor OVA-specific T cells with an activated phenotype)(CD45.2⁺ CD4⁺ CD62L^(lo)) present in the lymph node and BAL of the shamand allergen-challenged host is depicted in FIG. 25. The number ofinfiltrating inflammatory cells to the lungs of the mice is indicated inFIG. 26.

FIGS. 27-28. Priming of inflammatory allergen-specific T cells isreduced in CD38 deficient mice, even when the T cells are from normalanimals. The number of donor OVA-specific T cells (CD45.2⁺ CD4⁺) presentin the lymph node and BAL of sham and allergen-challenged hosts isdepicted in FIG. 27. The number of activated CD62Llo donor T cellspresent in the lymph nodes is also shown in FIG. 27. The number ofinfiltrating inflammatory cells to the lungs of the mice is indicated inFIG. 28. A representative H&E section of the lungs of OVA challenged WTor CD38 KO mice is also depicted in FIG. 28.

FIG. 29. Allergen-induced inflammatory responses in the lungs arereduced in CD38 deficient mice. CD38 deficient (KO) or normal C57BL/6(WT) mice were primed with ovalbumin (OVA) on day 0 or were inoculatedwith PBS. On day 42 post-immunization, animals were either leftuntreated (prime only) or were challenged with 10 μg OVA administeredintranasally I time/day for the next 7 days (prime+challenge group andchallenge only group). The lungs were isolated from all groups of miceone day after the last administration of OVA and were prepared forhistological examination. H&E stained paraffin sections of arepresentative animal from each group are shown.

FIG. 30. Diabetes onset is delayed in CD38 deficient mice. CD38deficient (KO) or normal BALB/c (WT) mice were injected withStreptozotocin. Blood glucose levels were measured 10 and 17 days afterthe last STZ injection.

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for modulating the migratoryactivity of cells involving the regulation of CD38 ADP-ribosyl cyclaseactivity. The invention is based on the discovery that granulocytes suchas neutrophils and eosinophils from CD38KO mice cannot be efficientlyrecruited to sites of inflammation and infection in the body. Theinvention is based on the discovery that although CD38 ADP-ribosylcyclase activity is not essential for the initial activation ofgranulocytes such as neutrophils, it is critically important inregulating neutrophil chemotaxis both in vivo and in vitro. Inparticular, cADPR, a product of CD38 ADP-ribosyl cyclase activity, isrequired to induce calcium release from calcium stores present withinneutrophils. The release of calcium from this specialized store isnecessary for activation and opening of plasma membrane channelsresulting in a capacitative influx of calcium that subsequently mediatesthe direct migration of neutrophils toward chemoattractants and/orinflammatory products.

The present invention encompasses screening assays designed for theidentification of modulators, such as agonists and antagonists, of CD38enzyme activity and/or modulators of cADPR dependent calcium responsesand chemotaxis. The invention further relates to the use of suchmodulators in the treatment of disorders based on the CD38 controlledmigratory activity of cells to chemoattractants and inflammatoryproducts. Such disorders include, but are not limited to, inflammation,ischemia, autoimmune disease, asthma, diabetes, arthritis, allergies,infections and organ transplant rejection.

The present invention also relates to the identification, isolation andcharacterization of the CD38 homologue, SM38, from the parasite S.mansoni. The invention encompasses screening assays to identify relatedenzymes in other pathogenic micro-organisms, such as helminths, as wellas compositions and assays to screen for compounds that modulate theactivity and expression of SM38. The invention further relates to theuse of such modulators to treat pathogenic disorders in animals andhumans infected with organisms expressing SM38 or structurally relatedmolecules.

Various aspects of the invention are described in greater detail in thesubsections below.

5.1 The SM38 Gene

The cDNA sequence and deduced amino acid sequence of S mansoni SM38 isshown in FIG. 13A (ATCC Deposit Nos: PTA-3780 (plasmid pCR2.1-TOPO in E.coli: SM38 5-18; PTA-3781 (plasmid SK in E. coli: SM38 LC12). The SM38cDNA was translated in all reading frames and an open reading frame of303 amino acids was identified. The initiation codon is located atnucleotide position 71 and the termination codon is found at nucleotideposition 981.

The SM38 nucleotide sequences of the invention include: (a) the DNAsequences shown in FIG. 13; (b) a nucleotide sequences that encodes theamino acid sequence shown in FIG. 13; (c) any nucleotide sequence that(i) hybridizes to the nucleotide sequence set forth in (a) or (b) understringent conditions, e.g., hybridization to filter-bound DNA in 0.5 MNaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., andwashing in 0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds., 1989,Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3)and (ii) encodes a functionally equivalent gene product; and (d) anynucleotide sequence that hybridizes to a DNA sequence that encodes theamino acid sequence shown in FIG. 13 under less stringent conditions,such as moderately stringent conditions, e.g., washing in 0.2×SSC/0.1%SDS at 42° C. (Ausubel et al., 1989 supra), yet which still encodes afunctionally equivalent SM38 gene product. Functional equivalents of theSM38 protein include naturally occurring SM38 present in species otherthan S. mansoni. The invention also includes degenerate variants ofsequences (a) through (d). The invention also includes nucleic acidmolecules, that may encode or act as SM38 antisense molecules, useful,for example, in SM38 gene regulation (for and/or as antisense primers inamplification reactions of SM38 gene nucleic acid sequences).

In addition to the SM38 nucleotide sequences described above, homologsof the SM38 gene present in other species can be identified and readilyisolated, without undue experimentation, by molecular biologicaltechniques well known in the art. For example, cDNA libraries, orgenomic DNA libraries derived from the organism of interest can bescreened by hybridization using the nucleotides described herein ashybridization or amplification probes.

The invention also encompasses nucleotide sequences that encode mutantSM38s, peptide fragments of the SM38, truncated SM38, and SM38 fusionproteins. These include, but are not limited to nucleotide sequencesencoding polypeptides or peptides corresponding to the cyclase domain ofSM38 or portions of this domain; truncated SM38s in which the domain isdeleted, e.g., a functional SM38 lacking all or a portion of the cyclaseregion. Certain of these truncated or mutant SM38 proteins may act asdominant-negative inhibitors of the native SM38 protein. Nucleotidesencoding fusion proteins may include but are not limited to full lengthSM38, truncated SM38 or peptide fragments of SM38 fused to an unrelatedprotein or peptide such as an enzyme, fluorescent protein, luminescentprotein, etc., which can be used as a marker.

SM38 nucleotide sequences may be isolated using a variety of differentmethods known to those skilled in the art. For example, a cDNA libraryconstructed using RNA from cells or tissue known to express SM38 can bescreened using a labeled SM38 probe. Alternatively, a genomic librarymay be screened to derive nucleic acid molecules encoding the SM38protein. Further, SM38 nucleic acid sequences may be derived byperforming PCR using two oligonucleotide primers designed on the basisof the SM38 nucleotide sequences disclosed herein. The template for thereaction may be cDNA obtained by reverse transcription of mRNA preparedfrom cell lines or tissue known to express SM38.

The invention also encompasses (a) DNA vectors that contain any of theforegoing SM38 sequences and/or their complements (i.e., antisense); (b)DNA expression vectors that contain any of the foregoing SM38 sequencesoperatively associated with a regulatory element that directs theexpression of the SM38 coding sequences; (c) genetically engineered hostcells that contain any of the foregoing SM38 sequences operativelyassociated with a regulatory element that directs the expression of theSM38 coding sequences in the host cell; and (d) transgenic mice or otherorganisms that contain any of the foregoing SM38 sequences. As usedherein, regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression.

5.1.2. SM38 Proteins and Polypeptides

SM38 protein, polypeptides and peptide fragments, mutated, truncated ordeleted forms of the SM38 and/or SM38 fusion proteins can be preparedfor a variety of uses, including but not limited to the generation ofantibodies, the identification of other cellular gene products involvedin the regulation of SM38 activity, and the screening for compounds thatcan be used to modulate the activity of SM38.

FIG. 13 shows the deduced amino acid sequence of the SM38 protein. TheSM38 amino acid sequences of the invention include the amino acidsequence shown in FIG. 13. Further, SM38s of other species areencompassed by the invention. In fact, any SM38 protein encoded by theSM38 nucleotide sequences described above is within the scope of theinvention.

The invention also encompasses proteins that are functionally equivalentto the SM38 encoded by the nucleotide sequences described in Section5.1, as judged by any of a number of criteria, including but not limitedto the ability to catalyze the production of the calcium mobilizingsecond messenger, cADPR and thereby regulate calcium response. Suchfunctionally equivalent SM38 proteins include but are not limited toproteins having additions or substitutions of amino acid residues withinthe amino acid sequence encoded by the SM38 nucleotide sequencesdescribed, above, in Section 5.1, but which result in a silent change,thus producing a functionally equivalent gene product.

Peptides corresponding to one or more domains of SM38 as well as fusionproteins in which the full length SM38, a SM38 peptide or a truncatedSM38 is fused to an unrelated protein are also within the scope of theinvention and can be designed on the basis of the SM38 nucleotide andSM38 amino acid sequences disclosed herein. Such fusion proteins includefusions to an enzyme, fluorescent protein, or luminescent protein whichprovide a marker function.

While the SM38 polypeptides and peptides can be chemically synthesized(e.g., see Creighton, 1983, Proteins: Structures and MolecularPrinciples, W.H. Freeman & Co., N.Y.), large polypeptides derived fromSM38 and the full length SM38 itself may be advantageously produced byrecombinant DNA technology using techniques well known in the art forexpressing a nucleic acid containing SM38 gene sequences and/or codingsequences. Such methods can be used to construct expression vectorscontaining the SM38 nucleotide sequences described in Section 5.1 andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. (See, forexample, the techniques described in Sambrook et al., 1989, supra, andAusubel et al., 1989, supra).

A variety of host-expression vector systems may be utilized to expressthe SM38 nucleotide sequences of the invention. Where the SM38 peptideor polypeptide is expressed as a soluble derivative and is not secreted,the peptide or polypeptide can be recovered from the host cell.Alternatively, where the SM38 peptide or polypeptide is secreted thepeptide or polypeptides may be recovered from the culture media.Purification or enrichment of the SM38 from such expression systems canbe accomplished using appropriate detergents and lipid micelles andmethods well known to those skilled in the art. Such engineered hostcells themselves may be used in situations where it is important notonly to retain the structural and functional characteristics of theSM38, but to assess biological activity, i.e., in drug screening assays.

The expression systems that may be used for purposes of the inventioninclude but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors containing SM38 nucleotide sequences; yeasttransformed with recombinant yeast expression vectors containing SM38nucleotide sequences or mammalian cell systems harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells or from mammalian viruses.

Appropriate expression systems can be chosen to ensure that the correctmodification, processing and sub-cellular localization of the SM38protein occurs. To this end, host cells which possess the ability toproperly modify and process the SM38 protein are preferred. Forlong-term, high yield production of recombinant SM38 protein, such asthat desired for development of cell lines for screening purposes,stable expression is preferred. Rather than using expression vectorswhich contain origins of replication, host cells can be transformed withDNA controlled by appropriate expression control elements and aselectable marker gene, i.e., tk, hgprt, dhfr, neo, and hygro gene, toname a few. Following the introduction of the foreign DNA, engineeredcells may be allowed to grow for 1-2 days in enriched media, and thenswitched to a selective media. Such engineered cell lines may beparticularly useful in screening and evaluation of compounds thatmodulate the endogenous activity of the SM38 gene product.

5.1.3 Transgenic Animals

The SM38 gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates,e.g., baboons, monkeys, and chimpanzees may be used to generate SM38transgenic animals.

Any technique known in the art may be used to introduce the SM38transgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell,56:313-321); electroporation of embryos (Lo, 1983, Mol. Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry theSM38 transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. Thetransgene may also be selectively introduced into and activated in aparticular cell type by following, for example, the teaching of Lasko etal., (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236).The regulatory sequences required for such a cell-type specificactivation will depend upon the particular cell type of interest, andwill be apparent to those of skill in the art. When it is desired thatthe SM38 transgene be integrated into the chromosomal site of theendogenous SM38 gene, gene targeting is preferred. Briefly, when such atechnique is to be utilized, vectors containing some nucleotidesequences homologous to the endogenous SM38 gene are designed for thepurpose of integrating, via homologous recombination with chromosomalsequences, into and disrupting the function of the nucleotide sequenceof the endogenous SM38 gene.

Once transgenic animals have been generated, the expression of therecombinant SM38 gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of SM38 gene-expressing tissue may also beevaluated immunocytochemically using antibodies specific for the SM38transgene product.

5.1.4. Antibodies to SM38 Proteins

Antibodies that specifically recognize one or more epitopes of SM38, orepitopes of conserved variants of SM38, or peptide fragments of SM38 arealso encompassed by the invention. Such antibodies include but are notlimited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention may be used, for example, in conjunctionwith compound screening schemes, as described, below, for the evaluationof the effect of test compounds on expression and/or activity of theSM38 gene product.

For production of antibodies, various host animals maybe immunized byinjection with a SM38 protein, or SM38 peptide. Such host animals mayinclude but are not limited to rabbits, mice, and rats, to name but afew. Various adjuvants may be used to increase the immunologicalresponse, depending on the host species, including but not limited toFreund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin,dinitrophenol, and potentially useful human adjuvants such as BCG(Bacille Calmette-Guerin) and Corynebacterium parvum.

Polyclonal antibodies comprising heterogeneous populations of antibodymolecules, may be derived from the sera of the immunized animals.Monoclonal antibodies may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclasses thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titres of Mabs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used(Morrison et al., 1984, Proc Nat'l. Acad. Sci., 81:6851-6855; Neubergeret al., 1984, Nature, 312: 604-608; Takeda et al. 1985, Nature 314:452-454), Alternatively, techniques developed for the production ofhumanized antibodies (U.S. Pat. No. 5,585,089) or single chainantibodies (U.S. Pat. No. 4,946,778 Bird, 1988, Science 242: 423-426;Huston et al., 1988, Proc. Nat'l. Acad. Sci. USA, 85: 5879-5883; andWard et al., 1989, Nature 334: 544-546) may be used to produceantibodies that specifically recognize one or more epitopes of SM38.

5.2. Screening Assays for Compounds Useful in Modulating the Activity ofCD38/SM38

The present invention relates to screening assay systems designed toidentify compounds or compositions that modulate CD38/SM38 enzymeactivity, cADPR mediated signal transduction, or CD38/SM38 geneexpression, or that cleave SM38 from a cell membrane, and thus, may beuseful for modulation of cell migration or treatment of infection. Asused herein, “CD38/SM38” specifies CD38 and/or SM38.

5.2.1. Recombinant Expression of CD38

For purposes of developing screening assays designed to identifycompounds or compositions that modulate CD38/SM38 activity it may benecessary to recombinantly express the CD38/SM38 proteins. The cDNAsequence and deduced amino acid sequence of CD38 has been characterizedfrom several species including human, marine and rat as described inJackson, D. G. et al., 1990, J. Immunol. 151:3111-3118; Koguma, T. etal., 1994, Biochim Biophys Acta 1224:160-162 and Harada N et al., 1993,J Immunol 151:3111-3118, incorporated herein by reference. In addition,the cDNA and deduced amino acid sequence of Shistosoma mansoni, asdescribed herein may be utilized to recombinantly express the CD 38homologue, SM38, protein.

CD38/SM38 nucleotide sequences may be isolated using a variety ofdifferent methods known to those skilled in the art. For example, a cDNAlibrary constructed using RNA from a tissue known to express CD38/SM38can be screened using a labeled CD38/SM38 probe. Alternatively, agenomic library may be screened to derive nucleic acid moleculesencoding the CD38/SM38 protein. Further, CD38/SM38 nucleic acidsequences may be derived by performing a polymerase chain reaction (PCR)using two oligonucleotide primers designed on the basis of knownCD38/SM38 nucleotide sequences. The template for the reaction may becDNA obtained by reverse transcription of mRNA prepared from cell linesor tissue known to express CD38/SM38.

CD38/SM38 protein, polypeptides and peptide fragments, mutated,truncated or deleted forms of CD38/SM38 and/or CD38/SM38 fusion proteinscan be prepared for a variety of uses, including but not limited to thegeneration of antibodies, the identification of other cellular geneproducts involved in the regulation of CD38/SM38 mediated cellmigration, and the screening for compounds that can be used to modulatecell migration. CD38/SM38 fusion proteins include fusions to an enzyme,fluorescent protein, a polypeptide tag or luminescent protein whichprovide a marker function.

While the CD38/SM38 polypeptides and peptides can be chemicallysynthesized (e.g., see Creighton, 1983, Proteins: Structures andMolecular Principles, W.H. Freeman & Co., N.Y.), large polypeptidesderived from CD38/SM38 and the full length CD38/SM38 itself may beadvantageously produced by recombinant DNA technology using techniqueswell known in the art for expressing a nucleic acid containing CD38/SM38gene sequences and/or coding sequences. Such methods can be used toconstruct expression vectors containing the CD38/SM38 nucleotidesequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.(See, for example, the techniques described in Sambrook et al., 1989,supra, and Ausubel et al., 1989, supra).

A variety of host-expression vector systems maybe utilized to expressthe CD38/SM38 nucleotide sequences. Where the CD38/SM38 peptide orpolypeptide is expressed as a soluble protein or derivative (e.g.,peptides corresponding to the intracellular or extracellular domain) andis not secreted, the peptide or polypeptide can be recovered from thehost cell. Alternatively, where the CD38 peptide or polypeptide issecreted the peptide or polypeptides maybe recovered from the culturemedia. However, the expression systems also include engineered hostcells that express CD38/SM38 or functional equivalents, anchored in thecell membrane. Purification or enrichment of the CD38/SM38 from suchexpression systems can be accomplished using appropriate detergents andlipid micelles and methods well known to those skilled in the art. Suchengineered host cells themselves may be used in situations where it isimportant not only to retain the structural and functionalcharacteristics of the CD38/SM38, but to assess biological activity,i.e., in drug screening assays.

The expression systems that may be used for purposes of the inventioninclude but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors containing CD38/SM38 nucleotide sequences; yeasttransformed with recombinant yeast expression vectors containingCD38/SM38 nucleotide sequences or mammalian, helminth or insect cellsystems harboring recombinant expression constructs containing promotersderived from the genome of mammalian, helminth or insect cells or frommammalian or insect viruses.

Appropriate expression systems can be chosen to ensure that the correctmodification, processing and sub-cellular localization of the CD38/SM38protein occurs. To this end, eukaryotic host cells which possess theability to properly modify and process the CD38/SM38 protein arepreferred. For long-term, high yield production of recombinant CD38/SM38protein, such as that desired for development of cell lines forscreening purposes, stable expression is preferred. Rather than usingexpression vectors which contain origins of replication, host cells canbe transformed with DNA controlled by appropriate expression controlelements and a selectable marker gene, i.e., tk, hgprt, dhfr, neo, andhygro gene, to name a few. Following the introduction of the foreignDNA, engineered cells may be allowed to grow for 1-2 days in enrichedmedia, and then switched to a selective media. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds that modulate the endogenous activity of the CD38/SM38 geneproducts.

5.2.2. Non-Cell Based Assays

In accordance with the invention, non-cell based assay systems may beused to identify compounds that interact with, i.e., bind to CD38, andregulate the enzymatic activity of CD38. Such compounds may act asantagonists or agonists of CD38 enzyme activity and maybe used toregulate cell migration including but not limited to hematopoieticallyderived cells. Additionally, such compounds may be used to regulate thegrowth, muscle contractility, differentiation, maturation andreproduction of pathogenic micro-organisms expressing SM38 orstructurally related homologues. Recombinant CD38/SM38, includingpeptides corresponding to different functional domains or CD38/SM38fusion proteins may be expressed and used in assays to identifycompounds that interact with CD38/SM38.

To this end, soluble CD38/SM38 maybe recombinantly expressed andutilized in non-cell based assays to identify compounds that bind toCD38/SM38. Recombinantly expressed CD38/SM38 polypeptides or fusionproteins containing one or more of the CD38/SM38 functional domains maybe prepared as described above, and used in the non-cell based screeningassays. For example, the full length CD38/SM38, or a soluble truncatedCD38/SM38, e.g., in which the one or more of the cytoplasmic andtransmembrane domains is deleted from the molecule, a peptidecorresponding to the extracellular domain, or a fusion proteincontaining the CD38/SM38 extracellular domain fused to a protein orpolypeptide that affords advantages in the assay system (e.g., labeling,isolation of the resulting complex, etc.) can be utilized. Wherecompounds that interact with the cytoplasmic domain are sought to beidentified, peptides corresponding to the CD38 cytoplasmic domain andfusion proteins containing the CD38 cytoplasmic domain can be used.

The CD38/SM38 protein may also be one which has been fully or partiallyisolated from cell membranes or from the cytosol of cells, or which maybe present as part of a crude or semi-purified extract. As anon-limiting example, the CD38 protein may be present in a preparationof cell membranes and the SM38 protein may be present in a preparationof cell cytosol. In particular embodiments of the invention, such cellmembranes may be prepared using methods known to those of skill in theart.

The principle of the assays used to identify compounds that bind toCD38/SM38 involves preparing a reaction mixture of the CD38/SM38 and thetest compound under conditions and for time sufficient to allow the twocomponents to interact and bind, thus forming a complex which can beremoved and/or detected in the reaction mixture. The identity of thebound test compound is then determined.

The screening assays are accomplished by any of a variety of commonlyknown methods. For example, one method to conduct such an assay involvesanchoring the CD38/SM38 protein, polypeptide, peptide, fusion protein orthe test substance onto a solid phase and detecting CD38/test compoundor SM38/test compound complexes anchored on the solid phase at the endof the reaction. In one embodiment of such a method, the CD38/SM38reactant is anchored onto a solid surface, and the test compound, whichis not anchored, may be labeled, either directly or indirectly.

In practice, microtitre plates conveniently can be utilized as the solidphase. The anchored component is immobilized by non-covalent or covalentattachments. The surfaces may be prepared in advance and stored. Inorder to conduct the assay, the non-immobilized component is added tothe coated surfaces containing the anchored component. After thereaction is completed, unreacted components are removed (e.g., bywashing) under conditions such that any complexes formed will remainimmobilized on the solid surface. The detection of complexes anchored onthe solid surface can be accomplished in a number of ways. Where thepreviously non-immobilized component is pre-labeled, the detection oflabel immobilized on the surface indicates that complexes were formed.Where the previously non-immobilized component is not pre-labeled, anindirect label can be used to detect complexes anchored on the solidsurface; e.g., using a labeled antibody specific for the previouslynon-immobilized component.

Alternatively, a reaction is conducted in a liquid phase, the reactionproducts separated from unreacted components using an immobilizedantibody specific for CD38/SM38 protein, fusion protein or the testcompound, and complexes detected using a labeled antibody specific forthe other component of the possible complex to detect anchoredcomplexes.

In accordance with the invention, non-cell based assays may also be usedto screen for compounds that directly inhibit or activate enzymaticactivities associated with CD38/SM38. Such activities include but arenot limited to ADP-ribosyl cyclase activity, transglycosidationactivity, and NAD+glycohydrolase activity. To this end, a reactionmixture of CD38/SM38 and a test compound is prepared in the presence ofsubstrate and the enzymatic activity of CD38/SM38 is compared to theactivity observed in the absence of test compound. Substrates that maybe used in the assays for detection of CD38/SM38 enzyme activity includebut are not limited to NAD+and NADP and labeled forms thereof.Additionally, derivatives of NAD such as Nicotinamide guaninedinucleotide (NOD) and Nicotinamide 1, N⁶-etheno-adenine dinucleotide(1,N⁶ etheno-NAD) may be used.

In non-limiting embodiments of the invention, a reaction mixture ofCD38/SM38, a test compound and substrate is prepared and the activity ofCD38/SM38 is compared to the activity observed in the absence of thetest compound wherein decrease in the level of CD38/SM38 enzyme activityin the presence of the test compound indicates that a CD38/SM38antagonist has been identified. Alternatively, a reaction mixture ofCD38/SM38, a test compound and substrate is prepared and the activity ofCD38/SM38 is compared to the activity observed in the absence of thetest compound wherein an increase in the level of CD38/SM38 enzymeactivity in the presence of the test compound indicates that a CD38/SM38agonist has been identified.

The enzymatic activity of CD38/SM38 may be detected in a variety ofdifferent ways. For example, levels of cyclic adenosine diphosphateribose (cADPR) adenosine diphosphate ribose (ADPR) and/or nicotinic acidadenine dinucleotide phosphate (NAADP) can be measured using highperformance liquid chromatography (HPLC) or thin layer chromatography(TLC) (Aarhus R et al., 1995, J. Biochem. Chem. 270:30327-30333;Muller-Steffner H M, J. Biol. Chem. 271:23967-23972; and Lund F E etal., 1999, J. Immunology 162:2693-2702; Higashida, H. et al., 1997, J.Biol. Chem. 272:3127-3177) in conjunction with the use of radio-labeledsubstrates such as NAD or NADP or NA. Additionally, radioimmunoassays(Takahashi K et al., 1995, FEBS Lett 371:204-208; Vu C Q et al., 1997,Biochem Biophys Res Commun 236:723-726; Vu et al., Adv Exp Med Biol419:381-388; and Graeff R M et al., 1997, Methods Enzymol 280:230-241),bioassays (Aarhus R et al., 1995, J Biol. Chem. 270:30327-30333; ClapperD L et al., J. Biol. Chem. 262:9561-9568; and-Lee et al., J. Biol. Chem.264:1608-1615) and/or fluorescent assays (Graeff R M et al., 1996,Biochem. 35:379-386; Graeff et al., 1994, J. Biol. Chem.269:30260-30267; and Gadangi P et al., 1996, J. Immunol. 156:1937-1941)may be used for measuring cADPR, ADPR or NAADP levels. In yet anotherembodiment of the invention, derivatives of NAD such as NGD(Nicotinamide guanine dinucleotide) and Nicotinamide 1,N⁶-etheno-adenine dinucleotide (1,N⁶ etheno-NAD) may be used to measureCD38/SM38 enzyme activity. When the 1,N⁶ etheno-NAD is hydrolysed byCD38, one of the resulting products will fluoresce (Muller et al., 1983,Biochem. J. 212:459-464; and Cockayne D et al., 1998, Blood92:1324-1333). When the analog NGD is cyclized through the ADP-ribosylactivity of CD38/SM38 the product forms a fluorescent compound that canbe detected by fluorimeter (Graeff et al., 1996, Biochem 35:379-386; andGraeff et al., 1994, J. Biol. Chem. 269:30260-30267).

In another embodiment of the invention, computer modeling and searchingtechnologies will permit identification of potential modulators ofCD38/SM38 enzyme activity. For example, based on the knowledge of theAplysia cyclase active site (Munshi C. et al., 199, J. Biol. Chem. 274:30770-30777) and the CD38 active site (Lund F E et al., 1999, J.Immunology 162:2693-2702; Munshi, C et al., 2000, J. Biol. Chem.275:21566-21571; Graeff R et al., 2001, J. Biol. Chem. 276:12169-12173)and the study of complexes between CD38/SM38 substrates and substrateanologs, potential modulators of CD38/SM38 activity may be identified.

The three dimensional geometric structure of the active site may bedetermined using known methods, including x-ray crystallography, whichcan determine a complete molecular structure (see, for example, Prasad GS et al., Nature Struc. Biol. 3:957-964 which describes the crystalstructure of Aplysia ADP ribosyl cyclase). On the other hand, solid orliquid phase NMR can be used to determine certain intramoleculardistances. Any other experimental method of structure determination canbe used to obtain the partial or complete geometric structure of theCD38 active site.

Having determined the structure of the CD38/SM38 active site, candidatemodulating compounds can be identified by searching databases containingcompounds along with information on their molecular structure. Such asearch seeks compounds having structures that match the determinedactive site structure and that interact with the groups defining theactive site. Such a search can be manual, but is preferably computerassisted. These compounds found from this search are potential CD38modulating compounds.

Alternatively, these methods can be used to identify improved modulatingcompounds from an already known modulating compounds. For example, anumber of compounds that modulate the enzyme activity of other enzymesthat utilize NADI/NADP as substrates (i.e., PARP family homologues) havealready been identified. The composition of the known compound can bemodified and the structural effects of modification can be determinedusing experimental and computer modeling methods applied to the newcomposition. The altered structure is then compared to the active sitestructure of the compound to determine if an improved fit or interactionresults. In this manner systematic variations in composition, such as byvarying side groups, can be quickly evaluated to obtain modifiedmodulating compounds or substrates of improved specificity or activity.

5.2.3. Cell Based Assays

In accordance with the invention, a cell based assay system can be usedto screen for compounds that modulate the activity of CD38/SM38. Inaccordance with the invention, a cell-based assay system can be used toscreen for compounds that modulate the activity of CD38 and thereby,modulate the chemoattractant induced Ca²⁺ influx and the migration ofcells. Additionally, this cell based system can be used to screen forcompounds that modulate the activity of SM38, and thereby, modulateintracellular calcium release and/or muscle contractility in cells. Tothis end, cells that endogenously express CD38/SM38 can be used toscreen for compounds. Such cells include, for example, neutrophils,lymphocytes, eosinophils, macrophages and dendritic cells. In addition,S. mansoni cells that express SM38, may be used to screen for compounds.Alternatively, cell lines, such as 293 cells, COS cells, CHO cells,fibroblasts, and the like, genetically engineered to express CD38/SM38can be used for screening purposes. For screens utilizing host cellsgenetically engineered to express a functional CD38 protein, it would bepreferred to use host cells that are capable of responding tochemoattractants or inflammatory stimuli. For screens utilizing hostcells genetically engineered to express SM38, it would be preferable touse cells of S mansoni origin that are capable of responding to avariety of stimuli such as acetylcholine or high concentrations of K+ toinduce muscle contraction. Further, ooyctes or liposomes engineered toexpress the CD38/SM38 protein may be used in assays developed toidentify modulators of CD38/SM38 activity.

The present invention provides methods for identifying compounds thatalter one of more of the enzymatic activities of CD38/SM38, includingbut not limited to, NAD glycohydrolase activity, ADP-ribosyl cyclaseactivity and/or transglycosidation (exchange) activity. Specifically,compounds may be identified that promote CD38/SM38 enzyme activities,i.e., agonists, or compounds that inhibit CD38/SM38 enzyme activities,i.e., antagonists. Compounds that inhibit CD38 enzyme activities will beinhibitory for chemoattractant induced calcium responses and cellmigration (FIG. 2). Compounds that activate CD38 enzyme activity willenhance chemoattractant induced calcium responses and cell migration.Compounds that either activate or inhibit SM38 enzyme activities willalter the viability or functional activities of pathogenic organismsexpressing SM38. Such compounds maybe compounds that interact with theactive site of CD38/SM38 thereby modulating enzyme activity, orcompounds that compete/facilitate substrate binding to CD38/SM38 orcompete/inhibit catalysis of substrate (FIG. 2). Alternatively,compounds may be identified that modulate the activity of proteins thatmodify the CD38/SM38 protein, i.e., phosphorylate, ribosylate, etc., andthereby regulate the activity of CD38 (FIG. 3). Such proteins includefor example, ADP-ribosyl transferases which ribosylate CD38/SM38 andrender CD38/SM38 enzymatically inactive. In addition, compounds may beidentified that regulate CD38/SM38 expression and thereby regulate thelevel of enzyme activity within a cell (FIG. 4).

The present invention provides for methods for identifying a compoundthat activates CD38/SM38 enzyme activity comprising (i) contacting acell expressing CD38/SM38 and chemoattractant receptors with a testcompound in the presence of substrate and measuring the level ofCD38/SM38 activity; (ii) in a separate experiment, contacting a cellexpressing CD38/SM38 protein and chemoattractant receptors with avehicle control in the presence of substrate and measuring the level ofCD38/SM38 activity where the conditions are essentially the same as inpart (i), and then (iii) comparing the level of CD38/SM38 activitymeasured in part (i) with the level of CD38/SM38 activity in part (ii),wherein an increased level of CD38/SM38 activity in the presence of thetest compound indicates that the test compound is a CD38/SM38 activator.

The present invention also provides for methods for identifying acompound that inhibits CD38/SM38 enzyme activity comprising (i)contacting a cell expressing CD38/SM38 and chemoattractant receptorswith a test compound in the presence of a chemoattractant and substrateand measuring the level of CD38/SM38 activity; (ii) in a separateexperiment, contacting a cell expressing CD38/SM38 and chemoattractantreceptors with a chemoattractant and substrate and measuring the levelof CD38/SM38 activity, where the conditions are essentially the same asin part (i) and then (iii) comparing the level of CD38/SM38 activitymeasured in part (i) with the level of CD38/SM38 activity in part (ii),wherein a decrease level of CD38/SM38 activity in the presence of thetest compound indicates that the test compound is a CD38/SM38 inhibitor.

Depending on the assays used to detect CD38/SM38 activity, the methodsdescribed above for identifying activators and inhibitors of CD38/SM38may include the presence or absence of a chemoattractant in steps (i)and (ii). For example, when assaying directly for CD38/SM38 ADP-ribosylcyclase activity or the production of CD38/SM38 metabolites, thepresence of a chemoattractant or the expression of a chemoattractantreceptor on the test cell may not be required. However, in instanceswhere, for example, chemotaxis or changes in intracellular calciumlevels are measured in CD-38-expressing cells it may be necessary toinclude chemoattractants. Alternatively, when muscle contractility orchanges in intracellular calcium levels are measured in SM38-expressingcells, it may be necessary to include stimulants to activate musclecontraction and/or calcium release including, but not limited to,acetylcholine, serotonin (Day et al., 1994, Paristol. 108:425-432),FMRF-amide related peptides (FaRPs) (Day et al., 1994, Paristol.109:455-459) or high K+ concentrations in the media (Day et al., 1993,Paristol. 106:471-477). Additionally, it will be necessary to performthese experiments with host cells that express the receptors specificfor the stimulants utilized. Those skilled in the art will be able todetermine operative and optimal assay conditions by employing routineexperimentation.

A “chemoattractant”, as defined herein, is a compound or molecularcomplex that induces the migration of cells via a mechanism that isdependent on the production of cADPR by CD38. An example of such achemoattractant includes, but is not limited to, fMet-leu-Phe (fMLP).Other chemoattractants that may be used include, eotaxin, GRO-1, IP-10,SDF-1, BLC, Rantes, MIP-1A, MCP-3, MIP3a, IL-8, CLS, ELC, Lymphotactin,PAF, Ltb4, complement c5a and histamine.

In utilizing the cell systems described above, such cell systems, thecells expressing the CD38/SM38 protein are exposed to a test compound orto vehicle controls e.g., placebos): After exposure, the cells can beassayed to measure the activity of CD38/SM38 or the activity of the CD38dependent signal transduction pathway itself can be assayed.

The ability of a test molecule to modulate the activity of CD38/SM38maybe measured using standard biochemical and physiological techniques.Responses such as activation or suppression of CD38/SM38 ADP-ribosylcyclase activity or the production of CD38/SM38 metabolites such ascADPR and/or NAADP can be measured. Levels of cADPR, ADPR and/or NAADPcan be measured using HPLC or TLC in conjunction with the use ofradio-labeled substrates such as NAD or NADP or NA. Additionally,radioimmunoassays, bioassays and/or fluorescent assays, such as thosediscussed in Section 5.1.1, supra, may be used for measuring cADPR orNAADP levels. In yet another embodiment of the invention, derivatives ofNAD such as NGD (Nicotinamide guanine dinucleotide) and Nicotinamide 1,N⁶-etheno-adenine dinucleotide (1,N⁶ etheno-NAD) may be used to measureCD38/SM38 activity.

Test compounds may also be assayed utilizing cell based calcium and/ormigration assays to identify compounds that are capable of inhibiting oractivating chemoattractant induced CD38 dependent calcium responses andcell migration. In non-limiting embodiments of the invention, changes inintracellular Ca²⁺ levels may be monitored by the fluorescence of Ca²⁺indicator dyes such as Indo, Fluo-3 and Fura-Red, etc. Further, changesin membrane potential resulting from modulation of the CD38/SM38 enzymeactivity can be measured using a voltage clamp or patch recordingmethods. Directed migration of cells may also be monitored by standardchemotaxis assays in modified Boyden chambers or on slides. Such assaysystems are described in further detail in the working example of thepresent specification (See, Example 6). Muscle contractility may also bemeasured by standard assays described in detail in the literature (forexample: (Day et al., 1994 Parasitology 109:455-9) and referencestherein).

After exposure to the test compound, or in the presence of a testcompound, cells can be stimulated with a chemoattractant such as fMLP ora muscle activator or constrictor, such as high K+ concentrations,acetylcholine, endothelin, etc., and changes in intracellular calciumlevels, cADPR or NAADP levels, muscle, contractility and/or cellmigration may be measured. These measurements will be compared to cellstreated with the vehicle control. Increased levels of intracellularCa²⁺, increased production of cADPR, increases in muscle contractilityand/or increased migration of cells toward a chemoattractant in thepresence of a test compound indicates that the compound acts as anagonists to increase the Ca2+ response increase muscle contractility andincrease chemoattractant induced CD38 dependent cell migration.Decreased levels of intracellular Ca2+, decreased production of cADPR,decreased muscle contractility and/or decreased migration of cellstoward a chemoattractant in the presence of a test compound indicatesthat the compound acts as an antagonist and inhibits the Ca2+ response,decreases muscle contractility and inhibits chemoattractant induced CD38dependent cell migration (see, for example, FIGS. 2 and 3).

In addition, the assays of the invention may be used to identifycompounds that (i) function as substrates of CD38/SM38 enzymaticactivity and are converted into agonists or antagonists of cADPRdependent Ca2+ signal transduction pathway (FIG. 5). A compound fittingthese specifications is described in further detail in the workingexample of the present specification (Example 6, FIG. 11).Alternatively, the assays of the invention may be used (ii) to identifycompounds that specifically interfere with the cADPR mediated Ca2+signal transduction pathways (FIG. 6). In a non-limiting embodiment ofthe invention, test compounds may include chemical derivatives of anyknown and unknown substrates of CD38/SM38 (for example, the substrateanalog 8-Br-βNAD is converted into the modified product 8-Br-cADPR whichacts as an antagonist of cADPR mediated Ca2+ signal transduction). Thetest substrate may be administered to cells expressing CD38/SM38 and theappropriate chemoattractant receptors in the presence of thechemoattractant or muscle stimulant. Conversion of the modified testsubstrate into a modified product that is capable of modulating theactivity of cADPR can be measured utilizing the methods described above.Test substrates may also be assayed to determine their effect on calciuminflux, muscle contractility and/or cell migration. Intracellular Ca2+accumulation and directed migration to a chemoattractant can be measuredin cells treated with the test substrate and the chemoattractant andcompared to cells receiving the non-modified substrate. i.e., NAD and achemoattractant. Compounds which are converted into modified products,i.e., 8-Br-cADPR, and competitively or non-competitively inhibit cADPRinduced calcium responses, muscle contractility or directed migrationwill be identified as antagonists of the cADPR Ca²⁺ signaling pathway,while compounds that are converted into modified products that arecompetitive or non-competitive agonists of the cADPR Ca²⁺ signalingpathway will be defined as agonists or activators.

In yet another embodiment of the invention, compounds that directlyalter (i.e., activate or inactivate) the activity of cADPR, i.e.,induced calcium release and cell migration, can be tested in assays.Such agonists or antagonists would be expected to modulate the influx ofCa2+ into the cell resulting in changes in the cell's migratory activityor ability to contract. Antagonists would have reduced Ca2+ responses,reduced contractility and/or reduced migration in the presence of achemoattractant. Examples of antagonists include, but are not limited to8-NH₂-cADPR, 8-Br-cADPR, 8-CH₃-cADPR, 8-OCH3-cADPR and7-Deaza-8-Br-cADPR. A compound fitting these specifications is describedin further detail in the working example of the present specification(Example 6, FIG. 10). Agonists would have increased Ca2+ responses,increased contractility and/or increased migration in the presence ofchemoattractants. Examples of agonists include but are not limited to2′-deoxy-cADPR, 3′-deoxy-cADPR and 2′-phospho-cADPR. Assays for directmeasurement of cAPDR activity include the bioassays such as thosedescribed by Howard et al. (1995, Science 262:1056); Galione et al.(1993, Nature 365:456-459) and Lee and Aarhus (1991, Cell Regulation2:203-209).

Further, the assays of invention may identify compounds that are capableof activating CD38/SM38 enzyme activity, i.e., agonists, but whichdesensitize the calcium pathway by depletion of intracellular calciumstores. Such desensitization may, in some instances, lead to inhibitionof cell migration or muscle contraction due to the depletion of calciumstores. Thus compounds may be identified that function as agonists inCD38/SM38 enzyme assays but function as antagonists in chemotaxis ormuscle contraction assays. Such assays and compounds are within thescope of the present invention.

5.2.4. Assay for Compounds that Regulate the Expression of CD38/SM38

In accordance with the invention, a cell based assay system can be usedto screen for compounds that modulate the expression of CD38/SM38 withina cell. Data described herein indicates that expression of SM38 isdevelopmentally regulated in S. mansoni. In particular, SM38 isexpressed during worm pairing and such expression is maintained in theadult worms. Such an expression pattern provides a target for compoundsthat modulate SM38 expression. Assays may be designed to screen forcompounds that regulate CD38/SM38 expression at either thetranscriptional or translational level. In one embodiment, DNA encodinga reporter molecule can be linked to a regulatory element of theCD38/SM38 gene and used in appropriate intact cells, cell extracts orlysates to identify compounds that modulate CD38/SM38 gene expression.Such reporter genes may include but are not limited to chloramphenicolacetyltransferase (CAT), luciferase, β-glucuronidase (GUS), growthhormone, or placental alkaline phosphatase (SEAP). Such constructs areintroduced into cells thereby providing a recombinant cell useful forscreening assays designed to identify modulators of CD38/SM38 geneexpression.

Following exposure of the cells to the test compound; the level ofreporter gene expression may be quantitated to determine the testcompound's ability to regulate CD38/SM38 expression. Alkalinephosphatase-assays are particularly useful in the practice of theinvention as the enzyme is secreted from the cell. Therefore, tissueculture supernatant may be assayed for secreted alkaline phosphatase. Inaddition, alkaline phosphatase activity may be measured by calorimetric,bioluminescent or chemiluminescent assays such as those described inBronstein, I. et al. (1994, Biotechniques 17: 172-177). Such assaysprovide a simple, sensitive easily automatable detection system forpharmaceutical screening.

To identify compounds that regulate CD38/SM38 translation, cells or invitro cell lysates containing CD38/SM38 transcripts maybe tested formodulation of CD38/SM38 mRNA translation. To assay for inhibitors ofCD38/SM38 translation, test compounds are assayed for their ability tomodulate the translation of CD38/SM38 mRNA in in vitro translationextracts.

In an embodiment of the invention, the level of CD38/SM38 expression canbe modulated using antisense, ribozyme, or RNAi approaches to inhibit orprevent translation of CD38/SM38 mRNA transcripts or triple helixapproaches to inhibit transcription of the CD38/SM38 gene. Suchapproaches may be utilized to treat disorders such as inflammation andallergies where inhibition of CD38/SM38 expression is designed toprevent hematopoietically-derived cell migration or inhibition of SM38is designed to alter S. mansoni physiology and pathogenesis.

Antisense and RNAi approaches involve the design of oligonucleotides(either DNA or RNA) that are complementary to CD38/SM38 mRNA. Theantisense or RNAi oligonucleotides will be targeted to the complementarymRNA transcripts and prevent translation. Absolute complementarity,although preferred, is not required. One skilled in the art canascertain a tolerable degree of mismatch by use of standard proceduresto determine the melting point of the hybridized complex.

In yet another embodiment of the invention, ribozyme molecules designedto catalytically cleave CD38/SM38 mRNA transcripts can also be used toprevent translation of CD38/SM38 mRNA and expression of CD38/SM38. (See,e.g., PCT International Publication WO90/11364, published Oct. 4, 1990;Sarver et al., 1990, Science 247:1222-1225). Alternatively, endogenousCD38/SM38 gene expression can be reduced by targetingdeoxyribonucleotide sequences complementary to the regulatory region ofthe CD38/SM38 gene (i.e., the CD38 promoter and or enhancers) to formtriple helical structures that prevent transcription of the CD38/SM38gene in targeted hematopoietically-derived cells in the body. (Seegenerally, Helene, C. et al., 1991, Anticancer Drug Des. 6:569-584 andMaher, L J, 1992, Bioassays 14:807-815).

The oligonucleotides of the invention, i.e., antisense, ribozyme andtriple helix forming oligonucleotides, may be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). Alternatively, recombinant expression vectors may be constructedto direct the expression of the oligonucleotides of the invention. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. In a specific embodiment, vectors such as viralvectors may be designed for gene therapy applications where the goal isin vivo expression of inhibitory oligonucleotides in targeted cells.

5.2.5. Assay for Compounds that Cleave the GPI-Anchored SM38 Protein

Data described herein (see Example 9) indicate that SM38 is expressed asa GPI-anchored protein on the outer tegument of adult S. mansoni worms.Thus, compounds having the ability to cleave SM38 from the outertegument, or membrane, of S. mansoni may be particularly useful in thetreatment of schistosomiasis. Assays may be designed to screen forcompounds that cleave the SM38 protein from the membrane, and suchassays are encompassed by the present invention. In one embodiment, acell-based assay is used to detect compounds that cleave the SM38protein from the membrane. By way of non-limiting example, cellsexpressing SM38 may be incubated in a supernatant containing a testcompound for a sufficient period of time for the test compound to cleavethe membrane-bound SM38. The supernatant can then be removed. Thepresence of SM38 in the supernatant can be detected in a number of ways,including but not limited to immunoassays, and would indicate that thetest compound has the ability to cleave SM38 from the cell membrane. Ina second embodiment, cells expressing SM38 are anchored to a solidsurface, and a test compound is applied for a sufficient period of timefor the test compound to cleave the membrane-bound SM38. The testcompound is then removed, such as by washing. The presence ofmembrane-bound SM38 may then be detected in a variety of ways,indicating a test compound's inability to cleave SM38 from the cellmembrane. SM38 may be detected by such means as contacting an antibodythat recognizes SM38 to the anchored cells. The antibody may be labeledfor easy detection.

5.2.6. Compounds that can be Screened in Accordance with the Invention

The assays described above can identify compounds which modulateCD38/SM38 activity. For example, compounds that affect CD38/SM38activity include but are not limited to compounds that bind toCD38/SM38, and either activate enzyme activities (agonists) or blockenzyme activities (antagonists). Alternatively, compounds may beidentified that do not bind directly to CD38/SM38 but are capable ofaltering CD38/SM38 enzyme activity by altering the activity of a proteinthat regulates CD38/SM38 enzyme activity (see, FIG. 3) Compounds thatare substrates of CD38/SM38 that are converted into modified productsthat activate or inhibit the cADPR Ca2+ signal transduction pathway, theADPR Ca2+ signaling pathway, or the NAADP signaling pathway can also beidentified by the screens of the invention. Compounds that directlyactivate or inhibit the cADPR Ca2+ signal transduction pathway in cellscan also be identified. Additionally, compounds that activate CD38/SM38enzyme activity resulting in desensitization of the calcium pathwaymaybe identified. Such desensitizing compounds would be expected toinhibit cell migration. Further, compounds that affect CD38/SM38 geneactivity (by affecting CD38/SM38 gene expression, including molecules,e.g., proteins or small organic molecules, that affect transcription orinterfere with splicing events so that expression of the full length orthe truncated form of the CD38/SM38 can be modulated) can be identifiedusing the screens of the invention.

The compounds which may be screened in accordance with the inventioninclude, but are not limited to, small organic or inorganic compounds,peptides, antibodies and fragments thereof, and other organic compoundse.g., peptidomimetics) that bind to CD38/SM38 and either mimic theactivity triggered by any of the known or unknown substrates ofCD38/SM38 (i.e., agonists) or inhibit the activity triggered by any ofthe known or unknown substrates of CD38/SM38 (i.e., antagonists).Compounds that bind to CD38/SM38 and either enhance CD38/SM38 enzymeactivities (i.e., ADP-ribosyl cyclase activity, NAD glycohydrolaseactivity, transglycosidation activity), i.e., agonists, or compoundsthat inhibit CD38/SM38 enzyme activities, i.e., antagonists, in thepresence or absence of the chemoattractant or muscle stimulant will beidentified. Compounds that bind to proteins that alter/modulate theenzyme activity of CD38/SM38 will be identified. Compounds that mimicnatural substrates, i.e., NAD(P) and are converted by CD38/SM38 enzymeactivities into products that act as agonists or antagonists of thecADPR induced calcium release pathway can be identified. Compounds thatdirectly activate or inhibit the cADPR Ca2+ signal transduction pathwayin cells can be identified.

Compounds may include, but are not limited to, peptides such as, forexample, soluble peptides, including but not limited to members ofrandom peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature354:82-84; Houghten, R. et al., 1991, Nature 354:84-86); andcombinatorial chemistry-derived molecular library made of D- and/orL-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate, directed phosphopeptidelibraries; (see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′)₂ and FAb expression library fragments, and epitope bindingfragments thereof), and small organic or inorganic molecules.

Other compounds which maybe screened in accordance with the inventioninclude but are not limited to small organic molecules that affect theexpression of the CD38/SM38 gene or some other gene involved in theCD38/SM38 signal transduction pathway (e.g., by interacting with theregulatory region or transcription factors involved in gene expression);or such compounds that affect the enzyme activities of the CD38/SM38 orthe activity of some other factor involved in modulating CD38/SM38enzyme activity, such as for example, a protein that ribosylatesCD38/SM38 and thereby inactivates CD38/SM38 enzyme activities.

5.3. Compositions Containing Modulators of CD38/SM38 and their Uses

The present invention provides for methods of modulating cell migrationcomprising contacting a cell expressing CD38 with an effective amount ofa CD38 modulating compound, such as a CD38 agonist or antagonistidentified using the assays as set forth in Section 5.1 supra.Additionally, the present invention provides for methods of modulatingcalcium responses and/or muscle contractility comprising contacting acell expressing SM38 with an effective amount of a SM38 modulatingcompound, such as a SM38 agonist or antagonist identified using theassays as set forth in Section 5.1 supra. An “effective amount” of theCD38/SM38 inhibitor, i.e., antagonist, is an amount that decreaseschemoattractant induced cell migration decreases intracellular calciumlevels, decreases muscle contraction and/or that is associated with adetectable decrease in CD38/SM38 enzyme activity as measured by one ofthe above assays. An “effective amount” of the CD38/SM38 activator,i.e., agonist, is an amount that subjectively increases chemoattractantinduced cell migration, increases intracellular calcium levels,increases muscle contraction and/or that is associated with a detectableincrease in CD38/SM38 enzyme activity as measured by one of the aboveassays. Compositions of the invention also include modified CD38/SM38substrates, modulators of CD38/SM38 expression and agonists/antagonistsof cADPR.

The present invention further provides methods of modulating cellmigration in a subject, comprising administering to the subject, acomposition comprising a compound that modulates CD38 enzyme activityidentified as set forth in Section 5.1 supra. The composition maycomprise an amount of CD38 enzyme activator or inhibitor, modulators ofCD38 expression, modified CD38 substrates, or directagonists/antagonists of cADPR controlled Ca2+ responses. Accordingly,the present invention provides for compositions comprising CD38activators and inhibitors.

The present invention provides for compositions comprising an effectiveamount of a compound capable of modulating the activity of CD38, theexpression of CD38 and/or the activity of cADPR thereby regulating themigratory activity of cells, and a pharmaceutically acceptable carrier.In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans. Theterm “carrier” refers to a diluent, adjuvant, excipient, or vehicle withwhich the therapeutic is administered. Examples of suitablepharmaceutical carriers are described in “Remington's Pharmaceuticalsciences” by E. W. Martin.

The invention provides for treatment or prevention of various diseasesand disorders associated with cell migration by administration of acompound that regulates the expression or activity of CD38. Suchcompounds include but are not limited to CD38 antibodies; CD38 antisensenucleic acids, CD38 agonists and antagonists (see, FIGS. 2-3), modifiedCD38 substrates (see, FIG. 5) and cADPR agonists and antagonists (see,FIG. 6). In a non-limiting embodiment of the invention, disordersassociated with hematopoietic derived cell migration are treated orprevented by administration of a compound that regulates CD38 activity.Such disorders include but are not limited to inflammation, ischemia,asthma, auto-immune disease, diabetes, allergies, infections, arthritisand organ transplant rejections.

The compounds of the invention are preferably tested in vitro, and thenin vivo for a desired therapeutic or prophylactic activity, prior to usein humans. For example, in vitro assays which can be used to determinewhether administration of a specific therapeutic is indicated, includein vitro cell culture assays in which cells expressing CD38 are exposedto or otherwise administered a therapeutic compound and the effect ofsuch a therapeutic upon CD38 activity is observed. In a specificembodiment of the invention the ability of a compound to regulate, i.e.,activate or inhibit cell migration may be assayed.

The present invention further provides methods of modulating the musclecontraction or other physiologic parameters in helminths such as S.mansoni by administering to helminth infected subject, a compositioncomprising a compound that modulates SM38 enzyme activity identified asset forth in Section 5.1 supra. The composition may comprise an amountof SM38 enzyme activator or inhibitor, modulators of SM38 expression,modified SM38 substrates, or direct agonists/antagonists of cADPRcontrolled Ca2+ responses. Accordingly, the present invention providesfor compositions comprising SM38 activators and inhibitors.

The present invention provides for compositions comprising an effectiveamount of a compound capable of modulating the activity of SM38, theexpression of SM38 and/or the activity of cADPR thereby regulating theactivity and viability of the parasite, and a pharmaceuticallyacceptable carrier. In a specific embodiment, the term “pharmaceuticallyacceptable” means approved by a regulatory agency of the Federal or astate government or listed in the U.S. Pharmacopeia or other generallyrecognized pharmacopeia for use in animals, and more particularly inhumans. The term “carrier” refers to a diluent, adjuvant, excipient, orvehicle with which the therapeutic is administered. Examples of suitablepharmaceutical carriers are described in “Remington's Pharmaceuticalsciences” by E. W. Martin.

The invention provides for treatment or prevention of various diseasesand disorders associated with helminth infections. Such compoundsinclude but are not limited to SM38 antibodies; SM38 antisense nucleicacids, SM38 RNAi molecules, SM38 agonists and antagonists (see, FIGS.2-3), modified SM38 substrates (see, FIG. 5) and cADPR agonists andantagonists (see, FIG. 6). In a non-limiting embodiment of theinvention, disorders associated with helminth infection are treated orprevented by administration of a compound that regulates SM38 activity.Such disorders include but are not limited to granuloma formation andfibrosis in the liver and lung.

The compounds of the invention are preferably tested in vitro, and thenin vivo for a desired therapeutic or prophylactic activity, prior to usein humans. For example, in vitro assays which can be used to determinewhether administration of a specific therapeutic is indicated, includein vitro cell culture assays in which cells expressing SM38 are exposedto or otherwise administered a therapeutic compound and the effect ofsuch a therapeutic upon SM38 activity is observed. In a specificembodiment of the invention the ability of a compound to regulate, i.e.,activate or inhibit muscle contractility or intracellular calciumaccumulation. Additionally, the compounds of the invention may beassayed for their effect on S. mansoni pathogenesis, growth,differentiation, and reproduction in a mouse model for S. mansoniinfection. Such assays would include the testing for effects onproliferation of parasites, maturation of female worms, quantity ofgranulomas in liver and lung, quantity of eggs in liver, lung bladderand intestines, quantity of worms in lung and liver and quantity ofmiracidia detected in urine and feces.

Additionally, the compounds of the invention may be assayed for theireffect on S. mansoni pathogenesis, growth, differentiation, andreproduction. Such compounds could be tested in a mouse model for S.mansoni infection. Such assays would include the testing for effects onproliferation of parasites, quantity of granulomas in liver and lung,quantity of eggs in liver, lung bladder and intestines and quantity ofmiracidia detected in urine and feces.

The invention provides methods of treatment and/or prophylaxis byadministration to a subject of an effective amount of a compound of theinvention. In a preferred aspect, the compound is substantiallypurified. The subject is preferably an animal, and is preferably amammal, and most preferably human.

Various delivery systems are known and can be used to administer acompound capable of regulating CD38 activity, cADPR, or CD38 expression,e.g., encapsulation in liposomes, microparticles, microcapsules,recombinant cells capable of expressing the compound, receptor-mediatedendocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432).Methods of introduction include but are not limited to intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, and oral routes. The compounds may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. Pulmonary administration can also be employed, by useof an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a specific embodiment, it may be desirable to administer thecompositions of the invention locally to a specific area of the body;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, topical application, e.g., in conjunctionwith a wound dressing after surgery, by injection, by means of acatheter, by means of a suppository, or by means of an implant, saidimplant being of a porous, non porous, or gelatinous material, includingmembranes, such as sialastic membranes, or fibers.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compoundcapable of regulating CD38 activity, cADPR activity or CD38 expressionand a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other Generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. The composition can be formulatedas a suppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carvers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the therapeutic compound,preferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Theformulation should suit the mode of administration.

The amount of the compound of the invention which will be effective inthe treatment of a particular disorder or condition will depend on thenature of the disorder or condition, and can be determined by standardclinical techniques. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachpatient's circumstances. Effective doses maybe extrapolated from doseresponse curves derived from in vitro or animal model test systems.Additionally, the administration of the compound could be combined withother known efficacious drugs if the in vitro and in vivo studiesindicate a synergistic or additive therapeutic effect when administeredin combination.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention, optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

5.4. Vaccine Directed to SM38

The SM38 protein, as well as polypeptide components, may be used invaccines against S. mansoni. The vaccines comprise an immunologicallyeffective amount of the immunogen, i.e., SM38, in a pharmaceuticallyacceptable carrier. The combined immunogen and carrier may be an aqueoussolution, emulsion, or suspension. An immunologically effective amountis determinable by means known in the art without undue experimentation,given the teachings contained herein. The carriers are known to thoseskilled in the art and include stabilizers, diluents, and buffers.Suitable stabilizers include carbohydrates, such as sorbitol, lactose,manitol, starch, sucrose, dextran, and glucose and proteins, such asalbumin or casein. Suitable diluents include saline, Hanks BalancedSalts, and Ringers solution. Suitable buffers include an alkali metalphosphate, an alkali metal carbonate, or an alkaline earth metalcarbonate. The vaccine may also contain one or more adjuvants to improveimmunogenicity. Suitable adjuvants include aluminum hydroxide, aluminumphosphate, or aluminum oxide or a composition that consists of a mineraloil, such as Marcol 52, or a vegetable oil and one or more emulsifyingagents.

The vaccines of the invention are prepared by techniques known to thoseskilled in the art, given the teachings contained herein. Generally, theimmunogens are mixed with the carrier to form a solution, suspension, oremulsion. One or more of the additives discussed above may be in thecarrier or may be added subsequently. The vaccine preparations may bedessicated, for example, by freeze drying for storage purposes. If so,they may be subsequently reconstituted into liquid vaccines by theaddition of an appropriate liquid carrier.

The vaccines are administered to humans or other mammals susceptible toS. mansoni infection, or related worm infections. They can beadministered in one or more doses. The vaccines may be administered byknown routes of administration for this type of vaccine. The preferredroutes are intramuscular or subcutaneous injection. Accordingly, theinvention also comprises a method for inducing an immune response toSM38 in a mammal in order to protect the mammal against infection by S.mansoni. The method comprises administering an immunologically effectiveamount of the immunogens of the invention to the host and, preferably,administering the vaccines of the invention to the host.

6. EXAMPLE Neutrophils Require CD38 for Chemotaxis, Capacitative Ca⁺Entry and Bacterial Clearance

The subsection below describes data demonstrating that calcium entry inchemoattractant activated neutrophils is controlled by cADPR, a productof the CD38 enzyme reaction. The capacitative calcium influx, controlledby the cADPR produced by CD38, is required for neutrophils to migrateefficiently to chemoattractants.

6.1. Materials and Methods 6.1.1. Mice

C57BL/6J×129 CD38KO F2 animals (Cockayne et al., 1998 Blood92:1324-1333) were backcrossed 6 generations (N6) to C57BL/6J and theninbred to produce homozygous congenic C57BL/6J.129 CD38KO mice.CD38-Rag-2 double KO (dKO) mice were produced by crossing C57BL/6J.129(N6) CD38KO mice with C57BL/6J.129 (N8) Rag-2 KO mice (Shin Kai et al.,1992 Cell 68:855-867) and then mating the offspring to obtain homozygousdouble KO animals. Bone marrow chimeric mice were produced bytransplanting 1×10⁷ whole bone marrow cells isolated from WT or CD38KOmice into lethally irradiated (950 rad) WT hosts. All mice were bred andmaintained in the Trudeau Institute Animal Breeding Facility.

6.1.2. cADPR Content Measurements

Mouse tissues were isolated from whole-body perfused WT or CD38KO miceand were flash frozen in liquid nitrogen. Bone marrow myeloid cells wereflushed from the tibias, and femurs of Rag-2KO or Rag-2-CD38 dKO mice.cADPR content in mouse tissues and bone marrow myeloid cells was thenmeasured as previously described (Vu et al., 1997 Biochem Biophys ResCommun 236:723-726).

S. pneumoniae infection. Mice were infected intra-tracheally with 100 or1000 CFU S. pneumoniae type 4 (Klein Strain) from American Type TissueCulture (Rockville, Md.). Blood, bronchial-aveolar lavage fluid (BAL)and lung tissue were collected from infected mice (Garvy et al., 1996Inflammation 20:499-512). Bacterial titers in lung homogenate and bloodwere calculated on a per lung basis or per ml of blood. BAL cells wereenumerated from cyto-centrifuge preparations.

6.1.3. In Vitro Chemotaxis Assays

Bone marrow neutrophils were purified (95% purity) by positive selectionusing biotinylated GR-1 (PharMingen) and MACS Streptavidin Microbeads(Miltenyi Biotec, Auburn Calif.). Chemotaxis assays (Falk et al., 1980J. Immuno. Methods 33:239-247) were performed using 24-well transwellplates with a 3 μm pore size polycarbonate filter (Costar, Cambridge,Mass.). Medium (HBSS+Ca2⁺+Mg2⁺), fMLP (1 μM, Sigma, St. Louis, Mo.), orIL-8 (100 nM, Sigma) was placed in the lower and/or upper chamber in acheckerboard format. 1×105 neutrophils were loaded in the upper chamberand the plates were incubated at 37° C. for 45 min. The transmigratedcells were collected from the lower chamber, fixed and counted on theflow cytometer (FACS Calibur, Becton Dickinson, San Jose Calif.). Todetermine the absolute number of cells in each sample, a standard numberof 20 μM size fluorescent microspheres (Polysciences, Inc. Warrington,Pa.) was added to each tube and counted along with the cells. The totalnumber of transmigrated cells=the number of counted neutrophils×totalnumber of beads/beads counted. In some experiments, neutrophils wereincubated in EGTA (2 mM) or pre-treated for 20 min with 8-Br-cADPR(25-100 μM, Sigma) or N(8-Br-A)D+(1.0 mM).

6.1.4. CD38 Expression

Bone marrow, blood or peritoneal cavity cells were isolated from WT orCD38KO mice and stained with anti-mouse GR-0.1 FITC, anti-mouse MAC-1 PEand anti-mouse CD38 APC (PharMingen, San Diego Calif.). Human peripheralblood neutrophils were isolated on a ficoll gradient and then stainedwith anti-hCD15-FITC (Becton Dickinson, San Jose Calif.) andanti-hCD38-Biotin (Caltag Laboratories, Burlingame Calif.). Mouse andhuman neutrophils were analyzed by flow cytometry, gating on the MAC-1⁺GR-1⁺ for mouse neutrophils and CD 15⁺ for human neutrophils. To inducean inflammatory response, mice were injected with 1 ml 3% thioglycollatemedium intra-peritoneally (Becton Dickinson, Cockeysville Md.). Theanimals were sacrificed 12 hrs post-injection, and the cellsinfiltrating the peritoneal cavity were collected.

6.1.4. Measurement of CD38 Cyclase Activity

Measurement of CD38 cyclase activity. 1×10⁶ purified bone marrowneutrophils were incubated for 20 min at 37° C. in 100 μl HBSS in a 96well microplate. NGD (40 μM) (Sigma) was added and the enzymaticconversion of NGD⁺ to cGDPR was measured fluorometrically (Graeff etal., 1994 J. biol. Chem. 269:30260-30267) over the next 10 minutes (415nm emission and 300 nm excitation).

6.1.5. RyR.-3 MRNA Expression in Neutrophils

cDNA was prepared from RNA isolated from purified bone marrowneutrophils or brain tissue. 30 cycles (annealing temperature 61° C.)RT-PCR was performed with 0.03-2 μg input cDNA and RyR-3 specificprimers (Guse et al., 1999 Nature 398:70-73).

Synthesis of N(S-Br-A)D⁺. N(8-Br-A)D⁺ was synthesized as previouslydescribed (Abdallah et al. 1975 Eur. J Biochem 50:475-481).

6.1.6. Intracellular Calcium Measurements

Purified bone marrow neutrophils were resuspended in cell loading media(HBSS with Ca2⁺ and Mg2⁺+1% FBS+4 mM probenecid) at 1×10⁷ cells/ml. Thecells were incubated at 37° C. for 30 min with the fluorescent dyesFluo-3 AM (4 μg/ml) and Fura Red AM (10 μg/ml) (Molecular Probes, EugeneOR) and then washed twice and resuspended in cell loading medium orcalcium-free medium at 1×10⁶ cells/ml. In some experiments, cells werepermeabilized in 5 μM digitonin in calcium-free media. In otherexperiments, cells were preincubated with EGTA (2 mM), 8-Br-cADPR(10-100 μM), ruthenium red (Sigma) or N(8-Br-A)D⁺ (1 mM) and thenstimulated with the carrier control (DMSO 0.01%), fMLP (1 μM), IL-8 (100nM), ryanodine (1 μM), cADPR (100 μM) or thapsigargin (1 μM). Theaccumulation of [Ca²+]i in individual cells was assessed by flowcytometry measuring the fluorescence emission of Fluo-3 in the FL-1channel and Fura-Red in the FL-3 channel. Data was analyzed using FlowJo3.2 (Tree Star, Inc. San Carlos, Calif.). The relative [Ca2⁺]i wasexpressed as the ratio between Fluo-3 and Fura Red mean fluorescenceintensity over time.

6.2. Results

CD38 is the primary ADP-ribosyl cyclase expressed in lymphoid tissues.To directly test the requirement for CD38 and cADPR in calcium-sensitiveimmunologic responses in vivo, CD38 knockout (CD38KO) mice wheregenerated (Cockkayne et al. 1998 Blood 92:1324-1333), To determinewhether CD38 is the primary cyclase expressed in mice, the cADPR contentin tissues and cells isolated from CD38KO and C57BL/6J wild-type (WT)mice were compared (Table 1).

TABLE 1 Comparison of cADPR content in tissues isolated from CD38KO andWT animals. cADPR content WT tissue cADPR content CD38KO Tissue (pmol/mgprotein) tissue (pmol/mg protein) Spleen 2.108 ± 0.334  (0.298 ± 0.091*Thymus 0.769 ± 0.182  0.335 ± 0.088** BM myeloid 0.633 ± 0.111  0.257 ±0.032* Lung 0.847 ± 0.213 0.480 ± 0.069 Kidney 0.488 ± 0.119 0.418 ±0.070 Heart 1.249 ± 0.324 1.014 ± 0.237 Brain 3.865 ± 0.866 3.127 ±0.316 Extracts were prepared from tissues isolated from 8-12 wk oldCD38KO or WT mice or from bone marrow (BM) myeloid cells isolated fromRag-2KO or Rag-2-CD38 double KO mice and were analyzed for cADPRcontent. Three separate purifications and analyses were performed ontissues isolated from 3 mice/analysis. *P-0.01, **P = 0.07; Anovaanalysis. Limit of detection, 0.2 pmol/mg protein.

WT tissues containing primarily lymphoid or myeloid cells, such asspleen, thymus and lymphoid deficient bone marrow (myeloid cells), hadeasily detectable levels of cADPR. In contrast, cADPR was not detectedin lymphoid or myeloid tissues isolated from CD38KO mice. However, thecADPR content of CD38KO tissues such as brain, kidney and heart wasnearly equivalent to the cADPR content of the same WT tissues. Thus,other unknown cyclases must be responsible for cADPR production inorgans such as brain and heart, however, CD38 is the predominantADP-ribosyl-cyclase expressed by myeloid and lymphoid cells.

CD38 deficient mice are more susceptible to bacterial infection. To testthe requirement for CD38 and cADPR in innate inflammatory immuneresponses, CD38KO and WT mice were infected with Streptococcuspneumoniae and assessed survival (FIG. 7A). It was observed that theLD50 for CD38 KO animals is at least 10-fold lower than for WT mice, as100 colony forming units (CFU) killed 50% of the CD38KO mice within 2.5days of infection, while 1000 CFU were required to kill 50% of the WTanimals in the same time period.

Since CD38 is expressed by the responding immunocytes and the bronchialepithelium (Fernandez J E et al., J. biol Reg Homeost Agents 12:81-91),WT or CD38KO bone marrow was transplanted into irradiated WT hosts totest whether CD38 expression in the lung and/or immune system wasnecessary for protection. The reconstituted chimeric animals possessedeither CD38+ or CD38-deficient bone-marrow derived cells, while allother cell types, including the bronchial epithelium, were of WT originin both groups of animals. The reconstituted mice were then infectedwith S pneumoniae and survival was monitored (FIG. 7B). Reconstitutedanimals receiving CD38KO bone marrow were much more susceptible toinfection compared to mice receiving WT bone marrow, indicating that theincreased susceptibility of CD38KO mice to S pneumoniae infection is dueto the loss of CD38 on bone marrow-derived lymphoid and/or myeloidcells.

To determine whether the increased susceptibility of CD38KO animals to Spneumoniae was due to an inability to restrain bacterial growth andspreading to systemic sites, CD38KO and WT mice were infected with 1000CFU of S. pneumoniae and bacterial titers were assessed in lung andblood 12 hours post-infection (FIG. 7C). The bacterial titer in thelungs of CD38KO mice was increased five-fold compared to WT controls.However, the bacterial burden in the blood of the CD38KO mice was200-500 times greater than in WT mice, indicating that the bacteriarapidly disseminate in CD38KO mice.

To determine whether myeloid or lymphoid cells were responsible for theincreased bacterial spreading, Rag-2 KO mice (Shin Kai, et al., 1992Cell 68:855-867) (which lack lymphocytes but can express CD38 on allmyeloid cells) and CD38-Rag-2 double knockout mice (which lacklymphocytes and cannot express CD38 on their myeloid cells) wereinfected with 1000 CFU S. pneumoniae and then bacterial titers weredetermined in lung and blood 12 hours later (FIG. 7C). The bacterialtiters in the lungs and blood of the lymphoid-deficient CD38-Rag-2double KO mice were as high as those seen in the CD38KO mice and weresignificantly increased when compared to Rag-2 KO or WT mice. Thus, CD38deficient myeloid cells are responsible for the increased susceptibilityof CD38KO mice to S. pneumoniae.

CD38 deficient neutrophils do not accumulate at sites of infection andinflammation. To test whether myeloid cells were appropriately recruitedto the lungs of S. pneumoniae-infected CD38KO animals, CD38KO and WTmice were infected and then the cells that were recruited to the lungairways after infection were enumerated. The total number of cells inthe airways of CD38KO and WT animals increased equivalently from 6 to 18hours post-infection (FIG. 8A). However, neutrophils were thepredominant cell type found in the lungs of WT animals 12-18 hourspost-infection, while the cellular infiltrate in the lungs of the CD38KOanimals was composed primarily of macrophages (FIG. 8B-C). Thus, CD38appears to be required for sustained recruitment of neutrophils to thesite of infection and inflammation.

CD38 deficient neutrophils make a defective chemotactic response to thechemoattractant fMLP. Neutrophils migrate to sites of infection inresponse to gradients of chemokines and chemoattractants that areproduced by the local cells and by the invading pathogen (Hub et al.1996 Chemoattractant Ligands and Their Receptors (ed. Horuk) 301-325(CRC Press, Boca Raton, Fla.); Servant G. et al., 2000 Science287:1037-1040; Gao, J. L., 1999 J. Exp. Med. 189:657-662).Chemoattractants rapidly activate neutrophils and induce randommigration (chemokinesis). If a chemotactic gradient exists, theactivated neutrophils polarize their leading edge toward the highestconcentration of the gradient and migrate directionally 16 (chemotaxis).It has been previously demonstrated that neutrophils home to sites ofinfection upon stimulation of their N-formylpeptide receptor (FPR) bybacterially-derived formylated such asformyl-methionyl-leucyl-phenyalanine (fMLP). To test whether CD38KOneutrophils were defective in their ability to chemotax to fMLP, theability of CD38KO and WT neutrophils to migrate by chemokinesis andchemotaxis in a transwell checkerboard assay was determined (Falk etal., 1980 J. Immunol. Methods 33:239-247) (FIG. 8D). When fMLP wasabsent from the top and bottom chamber, or when fMLP was placed only inthe top chamber, few (<2300 cells), but equivalent numbers, of theCD38KO and WT neutrophils migrated to the bottom chamber. When an equalconcentration of t1VILP was present in the top and bottom chamber(chemokinesis conditions), increased, but similar, numbers of WT andCD38KO neutrophils migrated to the bottom chamber, indicating thatactivation-induced chemokinesis to fMLP was equivalent between CD38KOand WT neutrophils. When fMLP was present in the bottom chamber only(chemotaxis conditions), the migration of WT neutrophils to the bottomchamber was further increased. However, CD38KO neutrophils migrated onlymarginally better in the presence of a chemotactic gradient than in theabsence of a fMLP gradient, indicating that CD38KO neutrophils can beactivated to migrate by bacterial chemoattractants but are unable tofollow the chemotactic gradient. To determine if this was a generalproperty of CD38KO neutrophils, the same experiments were performedusing the chemokine IL-8, which is a potent activator of neutrophils(Baggiolini et al., 1989 J. Clin. Invest 84:1045-1049). In contrast towhat was observed with fMLP, the IL-8-induced chemotaxis of CD38KO andWT neutrophils was equivalent (FIG. 8D). Thus, these data indicate thatCD38KO neutrophils make defective chemotactic responses to some, but notall, chemoattractants.

CD38 is expressed and enzymatically active on neutrophils. Since CD38KOneutrophils appear to have an intrinsic defect in chemotaxis, CD38expression and enzyme activity on mouse and human neutrophils wasdetermined. Neutrophils isolated from the bone marrow and blood of WTmice clearly expressed CD38 (FIG. 9A), and likewise, human peripheralblood neutrophils also expressed CD38 (FIG. 9B). Interestingly, when WTmice were injected intraperitoneally with the inflammatory agent,thioglycollate, CD38 expression increased significantly on theneutrophils isolated from the blood and peritoneal cavity (FIG. 9D).Next, to test whether CD38-expressing neutrophils can catalyze thecyclase reaction, WT and CD38KO neutrophils were incubated with theNAD+analogue, nicotinamide guanine dinucleotide (NGD), and then measuredthe cyclization of NGD into the fluorescent compound cyclic GDP-ribose(Graeff et al., 1994 J. Biol. Chem. 269:30260-30267) (cGDPR). As shownin FIG. 9C, WT neutrophils, but not CD38KO neutrophils, produced cGDPRrapidly upon incubation with NGD, indicating that CD38-expressingneutrophils are competent to produce cyclic nucleotides.

cADPR and ryanodine induce intracellular calcium release in neutrophils.Since cADPR induces intracellular calcium release through ryanodinereceptor (RyR) gated stores (Galione et al. 1991 Science 253:1143-1146),it was tested whether the RyR/cADPR calcium signaling pathway wasfunctional in neutrophils. RT-PCR analysis showed that neutrophilsexpress mRNA for RyR3 (Sorrentino, V. et al., 1993 TIPS 14:98-103;Hakamata Y. et al., 1992 FEBS Lett 312:229-235), although at levels muchlower than seen in the brain (FIG. 9D). To test whether the RyRsexpressed by neutrophils were functional, intracellular calcium levels([Ca2⁺]i) were measured in neutrophils that were permeabilized incalcium-free buffer and then stimulated with ryanodine (FIG. 9E). Asmall, but reproducible, increase in [Ca2⁺] in ryanodine-stimulatedneutrophils that could be blocked by the RyR inhibitor, ruthenium redwas observed (Galione et al. 1991 Science 253:1143-1146). Next, to testwhether cADPR could induce intracellular calcium release in neutrophils,neutrophils were permalized in calcium-free buffer and then stimulatedthe cells with purified cADPR (FIG. 9F). A small, but easily detectable,rise in intracellular free calcium was observed. No calcium release wasobserved when the cADPR was first hydrolyzed by heat inactivation (Leeet al., 1989 J. Biol. Chem. 264:1608-1615) or when the cells werepre-treated with 8-Br-cADPR, an inactive analogue of cADPR thatcompetitively antagonizes cADPR binding to RyRs (Guse et al., 1994 Annu.Rev. Immunol 12:593-633). The specificity of the antagonist, 8-Br-cADPR,for cADPR mediated calcium release was further demonstrated by showingthat 8-Br-cADPR was unable to block the accumulation of intracellularfree calcium mediated by thapsigargin (FIG. 9G). Together, the datademonstrate that intracellular calcium can be released through RyR andcADPR-mediated mechanism in neutrophils.

CD38 catalyzed cADPR is required for extracellular calcium influx infMLP-activated neutrophils. Signaling through chemokine/chemoattractantG-protein coupled receptors such as FPR and the IL-8 receptors resultsin increased [Ca2⁺]i due to a combination of intracellular calciumrelease and extracellular calcium influx (Murphy, P. M., 1994 Annu. Rev.Immunol 12:593-633; Demaurex N. et al., 1994 Biochem J. 297:595-601;Schorr W. et al., 1999 Eur. J. Immunol. 29:897-904: Lew et al., 1989Eur. J. Clin. Invest. 19:338-346). Since CD38KO neutrophils weredefective in chemotaxis assays to fMLP and lacked the ability to producethe calcium mobilizing metabolite, cADPR, it was hypothesized thatcalcium mobilization in response to fMLP would be deficient in CD38KOneutrophils. To test this, CD38KO or WT neutrophils were stimulated withfMLP or IT-8 in calcium-free media and intracellular calcium release wasmeasured (FIG. 10A). An immediate sharp rise in intracellular calciumwas observed that gradually declined over next 5 minutes infMLP-stimulated WT neutrophils. In contrast, in fMLP-stimulated CD38KOcells, the magnitude of [Ca2⁺]i after fMLP stimulation was reduced byapproximately 20% and the [Ca2⁺]i declined to baseline at least 2minutes earlier. Unlike the reduced [Ca2⁺]i found in fMLP-stimulatedCD38KO neutrophils, the [Ca2⁺]I of IL-8 stimulated CD38KO and WTneutrophils was identical. Thus, these data suggested that CD38 may benecessary for optimal intracellular calcium release after fMLP, but notIL-8, stimulation.

Next, to assess whether CD38 was required for extracellular calciuminflux, stimulated CD38KO or WT neutrophils were stimulated with fMLP orIL-8 in calcium-containing media (FIG. 10B). When we added fMLP to WTneutrophils, a rapid increase in [Ca2⁺]i, due to intracellular calciumrelease was observed, as well as a second extended, increase in [Ca2⁺]i,due to extracellular calcium influx. In striking contrast, the calciuminflux phase of the response was essentially ablated in thefMLP-stimulated CD38KO neutrophils. Interestingly, when WT and CD38KOneutrophils were stimulated with IL-8 in calcium containing media, itwas found that IL-8 induced a equivalent immediate increase in [Ca2⁺]ithat rapidly declined to baseline levels in both WT and CD38KOneutrophils, indicating that IL-8 did not induce extracellular calciuminflux in either WT or CD38KO neutrophils.

To determine whether cADPR regulates calcium mobilization in fMLPstimulated neutrophils, CD38KO and WT neutrophils were preincubated withincreasing concentrations of the cADPR antagonist, 8-Br-cADPR, and thenstimulated with fMLP or IL-8 (FIG. 10C). When 8-Br-cADPR-treated WTcells were stimulated with fMLP, the release of intracellular calcium aswell as the influx of extracellular calcium was reduced in adose-dependent fashion to the levels seen in CD38KO cells. In contrast,addition of 8-Br-cADPR to IL-8 stimulated neutrophils had absolutely noeffect on the [Ca2⁺]i of either WT or CD38KO neutrophils. Together,these data indicate that CD38-produced cADPR regulates intracellularcalcium release and extracellular calcium influx in response to fMLP,and that neither CD38 nor cADPR are necessary for calcium mobilizationin IL-8 stimulated neutrophils.

CD38 catalyzed cADPR is required for neutrophil chemotaxis to fMLP butnot IL-8. To test whether cADPR-mediated calcium mobilization isrequired for chemotaxis to AMP, WT neutrophils were preincubated witheither EGTA or 8-Br-cADPR and then chemotaxis to fMLP or IL-8 in acheckerboard chemotaxis assay was measured (FIG. 10D). When WTneutrophils (no pre-treatment) were incubated with media in the topchamber and fMLP or IL-8 in the bottom chamber, the cells efficientlymigrated to the bottom chamber. However, if the extracellular calciumwas chelated with EGTA or if the cells were pre-treated with the cADPRantagonist, 8-Br-cADPR, chemotaxis of the WT neutrophils to fMLP wasreduced by more than 80%. Importantly, EGTA or 8-Br-cADPR treatment hadabsolutely no effect on the ability of neutrophils to chemotax to IL-8.Thus, extracellular calcium influx, regulated by cADPR-mediatedintracellular calcium release, is necessary for EMLP-induced chemotaxisof neutrophils.

An analogue of NAD+inhibits neutrophil chemotaxis to fMLP, but not IL-8,in a CD38-dependent fashion. Since CD38 catalyzed cADPR appearednecessary for neutrophil chemotaxis to fMLP, it was predicted thatchemotaxis could be inhibited by treating neutrophils with NAD⁺analogies that could be converted by CD38 into antagonists of the cADPRsignaling pathway. To test this prediction, neutrophils with pretreatedwith nicotinamide 8-bromoadenine dinucleotide (N(S-Br-A)D⁺), a substratethat can be converted by CD38 into 8-Br-cADPR, the cADPR antagonist thatwas used in our earlier experiments. To first test whether N(8-Br-A)D⁺altered extracellular calcium influx in flVILP-activated neutrophils, WTneutrophils were pretreated with N(8-Br-A)D⁺, the cells were stimulatedwith AMP and then [Ca2⁺]i was measured (FIG. 11A). N(8-Br-A)D⁺pre-treatment inhibited the entry of extracellular calcium infMLP-treated neutrophils. Next, WT and CD38KO neutrophils werepretreated with N(8-Br-A)D⁺ or left in media alone, followed by testingfor their ability to chemotax to fMLP (FIG. 11B) or IL-8 (FIG. 11C).Untreated WT neutrophils chemotaxed to both fMLP and IL-8, whileuntreated CD38KO neutrophils could not chemotax to fMLP, but couldchemotax to IL-8. Interestingly, pre-treatment of WT neutrophils withN(8-Br-A)D⁺ severely reduced neutrophil chemotaxis to fMLP but had noeffect on their ability to chemotax to IL-8. Pre-treatment of the CD38KOneutrophils with N(8-Br-A)D⁺ had no effect on the chemotaxis of theCD38KO neutrophils to either flVILP or IL-8, indicating that theN(8-Br-A)D⁺ induced inhibition of fMLP-mediated chemotaxis was CD38dependent. Together, the data demonstrate that NAD⁺ analogues canregulate calcium responses and chemotaxis of neutrophils in aCD38-dependent fashion.

7. EXAMPLE Mouse Model of Allergic Lung Disease and Role of CD38

The subsection below describes data demonstrating that CD38-deficienteosinophils are unable to be recruited to the site of airwayinflammation induced by allergens.

7.1. Materials and Methods

OVA priming and sensitization. C57BL/6 WT mice were immunizedintraperitoneally with 20 μg chicken ovalbutnin (OVA) adsorbed to alum.Immunized mice were sacrificed 30 days post-immunization and theOVA-primed CD4 T cells were purified from the spleen using MACS magneticbeads that were directly conjugated with anti-CD4. Naive CD4 T cellswere purified using anti-CD4 conjugated MACS beads from unimmunizedC57BL/6 WT mice. Naive or OVA-primed T cells were injected intravenouslyinto either C57BL/6 WT or CD38KO recipients at 1×10⁷ CD4 T cells permouse to generate 4 groups of 10 mice each indicated in FIGS. 11A and B.Recipient mice were then sensitized intratracheally with 10 μg OVA inPBS on each of 7 consecutive days immediately following T cell transfer.Mice were sacrificed on the eighth day after T cell transfer, andinfiltrating cells were removed from the airways and alveoli of thelungs by broncheoalveolar lavage as described in Section 6.2.2, supra.Total cells were then enumerated by counting on a hemocytometer anddifferential cell counts were performed by centrifuging cells on to aglass slide, staining with Diff-Quick and identifying at least 200 cellsper slide at 400×.

7.2. Results

To determine if CD38 controls the recruitment of cells other thanneutrophils to the lung, a mouse model of allergic lung disease thatmimics many of the properties of human asthma was used (Lloyd C M etal., 2001, Adv. Immunol. 77:263-295). An important component a asthma isairway inflammation, which is thought to be induced or exacerbated bythe activities of eosinophils that have been recruited to the lung.Although eosinophils are primarily responsible for the pathology ofasthma (Broide, D H et al., 1991, J. Allergy Clin. Immunol. 88:637-648),their recruitment and function appears to be controlled by T cells thathave been primed to allergenic antigens (Gavett et al., 1994, Respir.Cell Mol. Biol. 10:587-593). Such T cells often produce type 2cytokines, such as IL-4, IL-5 and IL-13, as well as chemokines likeeotaxin (Cohn, L. et al., 1988, J. Immunol. 161:3813-3816; Drazen J M etal., 1996, J. Exper Med. 183:1-5). To examine the ability of CD38 toregulate eosinophil recruitment independently of any effects of CD38 onT cell activation, WT mice were immunized with the antigen OVA. After 30days, CD4 T cells from these OVA-primed mice were transferred to eitherWT or CD38KO recipients. As a control, naive CD4 T cells weretransferred from unimmunized WT mice to either WT or CD38KO recipients.Recipient mice were then sensitized intratracheally with 10 μg of OVA inPBS on each of eight consecutive days immediately following T celltransfer. Mice were sacrificed on the ninth day after T cell transferand the cells in the airways of the lungs were enumerated.

As seen in FIG. 12A, substantial numbers of neutrophils were recruitedto the airways of WT mice regardless of whether they received naive orprimed CD4 T cells. In contrast, although CD38KO trice that receivedprimed T cells did have significantly more neutrophils in the airwaysthan CD38KO mice that received naive T cells, relatively few neutrophilswere recruited to the airways of CD38KO mice compared to the airways ofWT mice. Thus, neutrophil recruitment to the lung in a model of allergicairway disease is also dependent on the expression of CD38.

Strikingly, the recruitment of eosinophils to the airways ofOVA-sensitized mice was dependent on the presence of both primed CD4 Tcells and the expression of CD38. As seen in FIG. 12B there was a30-fold reduction in the numbers of eosinophils recruited to the lungsof CD38 K0 mice that had received primed CD4 T cells relative to that inWT mice that had received primed CD4 T cells and a 10-fold reduction inthe numbers of eosinophils recruited to the airways of CD38KO mice thatreceived naive CD4 T cells relative to that in WT mice that had receivednaive CD4 T cells. Therefore, the recruitment of eosinophils to the lungin a model of allergic airway disease is also dependent on CD38.

8. EXAMPLE Cloning of S. mansoni CD38 Homologue

The subsection below describes the cloning and sequencing of a S.mansoni CD38 homologue referred to as SM38. Helminths, such as S.mansoni, are broadly defined as worm parasites that infect and can causepathogenesis in most invertebrates, vertebrates and plant species. Thegenus Schistosoma consists of parasitic flatworms whose definitivehabitat is the bloodstream of warm-blooded vertebrates. Four species ofSchistosoma, including S. mansoni cause disease in 200-400 millionhumans per year and kill up to 1 million people each year (WHO, 1996).Additionally, at least two Schistosoma species infect domesticatedcattle and sheep causing serious economic losses. Thus, it would bebeneficial to develop effective antibiotic drugs that could be used totreat infected humans and/or animals. The pathogenesis of Schistosomainfection is caused mainly by the deposition of eggs by the mature worminto various tissues and organs of humans and animals where granulomasthen form leading to fibrosis and tissue damage. However, the cercariae(immature worm) and fully mature worm also release a number of proteinsand lipid mediators that can also induce an immune inflammatory response(Fusco, A C et al., 1991, J. Paristol. 77:649-657). The treatment ofchoice in schistosomaisis is the drug praziquantel which appears toinduce calcium influx across the tegument of the worm causing immediatemuscle contraction and paralysis (Kohn, A B et al., 2001, J. Biol. Chem276:36873-36876). Thus, drugs that modulate schistosome calciumresponses, particularly within the muscle, might be effective in thetreatment of this disease.

8.1. Materials and Methods 8.1.1. Cloning of Shistosoma mansoni CD38Homologue

Primers were made corresponding to the EST sequence found in Genbankaccession #AW017229. (5′ primer: acatctttgtggtactgaatggctcgg and 3′primer: tgagtaatgtctcgacgtttgacctcg). S. mansoni cDNA libraries wereobtained from Dr. Philip LoVerde (SUNY, Buffalo), and were subjected toPCR using the primers indicated above. The library (1-20 μl) and dH20were heated to 70° C. for 10 minutes and were then combined with theremainder of the PCR reagents and cycled. The cycles were: 95° C. 5minutes, 1 ‘cycle, followed by 95° C. 1 minute, 65° C. 1 minute and 72°C. 2 minutes for 35 cycles followed by 1 cycle at 72° C. for 5 minutes.The expected 330 by band corresponding to EST AW017229 was isolated,TOPO cloned, and then used as a probe to screen 250,000 plaques from theS. mansoni cDNA library. Five positives were isolated and then subjectedto 3 more rounds of screening in order to produce plaque pure clones.All five clones were fully sequenced on both strands. The nucleotidesequence and amino acid translation of four of the clones were identical(referred to as SM38). The stop codon and polyadenylation sites wereidentified in all of the SM38 clones, but the initiation methionine wasnot present in any of the clones. To obtain the 5′ end of the SM38 gene,a single primer extension approach (NAR, 1994, vol 22, No. 16, p3427-3428) was utilized. A first round of PCR was performed using anexternal SM38 primer (5′ catcgaataaccctgatttcataacac) and the universalreverse primer for Bluescript. Two μL of this reaction was thensubjected to PCR using an internal nested SM38 primer (5′gataaagtaagaactcgtgcc) and the universal reverse primer. A 200 and a 300by band were identified from this reaction and were directly sequenced.The sequence obtained overlapped 124 by with the 5′ end of the SM38clones and included an additional 153 by of sequence, however the nostop codon was detected, indicating that we still did not have the 5′end of the gene. Therefore, classic 5″RACE (PNAS vol 85 pp 8998-9002,December 1998) was performed using cDNA prepared from RNA isolated fromadult S. mansoni worms (RNA provided by Dr. P. LoVerde, SUNY Buffalo).10× Taq buffer, dNTP's, cDNA and Expand High Fidelity Taq were combinedwith the dT-AP primer (see ref. For details) and cycled for 5 minutes at95° C. followed by 2 minutes at 50° C. and 40 minutes at 72° C. Afterthis 40 minute incubation the 5′ external SM38 primer (see above) and APprimers were added and cycled for 35 cycles under the conditions: 95° C.for 15 sec, 47° C. for 30 sec, 72° C. for 2 minutes followed by a 5minute extension at 720C. The reactions were run on a 1.5% agarose geland a 300 by band was isolated using Qiagen Gel Kit. The 5′ RACE productwas directly sequenced with the AP and 5′ external SM38 primer. Twopotential initiation methionines were identified in the sequence and twostop codons were found 13-19 amino acids upstream of the methionineresidues. The RACE product was subsequently cloned. All three clonescontaining SM38 sequence (Two PCR generated clones and one clone fromthe S. mansoni cDNA library) were contiguous and overlapping. Whenassembled, the SM38 sequence included 1049 by of sequence including 5′untranslated sequence, two potential initiation methionines, an openreading frame encoding a 303 amino acid protein, a stop codon, 3′untranslated sequence and a poly-adenylation site.

8.2. Results

Since drugs that modulate calcium responses in the muscle fibers ofSchistosomes appear to be effective anti-helminth reagents (Kohn et al,2001, J. Biol. Chem. 276:36873-36876), we set out to identify specificcalcium modulating targets of Schistosomes. It has been recently shownthat Schistosomes express Ryanodine Receptors (RyR) within their musclefibers (Day et al., 2000, Parasitol. 120:417-420; Silva et al., 1998,Biochem. Pharmacol. 56:997-1003). Agonists of RyRs expressed invertebrate smooth and skeletal muscle are known to regulateintracellular calcium release, voltage gated calcium influx and musclecontractility. Interestingly, S. mansoni muscle fibers treated with RyRagonists such as caffeine induced the release of intracellular calciumand induced contraction of the muscle fiber. Although drugs such ascaffeine can modulate RyR-dependent calcium responses, the physiologicalmodulator of RyRs, at least in vertebrate muscle fibers is cyclicADP-ribose (cADPR). cADPR is a known calcium mobilizing metabolite thatis produced by ADP-ribosyl cyclase enzymes such as the mammalian CD38protein and the invertebrate Aplysia cyclase enzyme. To determinewhether Schistosomes express an enzyme capable of producing the calciummobilizing second messenger, cADPR, a search was performed of thepublicly available EST sequences looking for Schistosome sequences thatwhen translated would have homology to the mammalian CD38 and Aplysiaenzymes. Three EST sequences (EST A1067047, EST AW017229 and EST N20756)were identified that could be assembled into a contiguous andoverlapping sequence (FIG. 13). This assembled sequence shared limitedbut significant homology with both CD38 and the Aplysia cyclase enzymes.

To determine whether the assembled ESTs actually represented anauthentic cDNA, primers were prepared from the sequence of EST AW017229and performed PCR on a S. mansoni cDNA library. A 330 base pair fragmentwas isolated from the PCR reaction and was sequenced. As expected thesequence of the fragment matched that of the EST. The fragment was thenused as a probe to screen 250,000 plaques from the S. mansoni cDNAlibrary. Five independent plaques which hybridized to the EST probe wereisolated, plaque purified and sequenced on both DNA strands. Thesequence information was then used to design additional primers toisolate the 5′ end of the cDNA (see methods). The complete cDNA sequenceisolated from the S. mansoni library was then assemble and compared tothe ESTs. The alignment, shown in FIG. 13, indicates that the contiguousassembly of the EST sequences was correct and that the cloned cDNA(referred to as SM38) included at least an additional 421 base pairs ofsequence not found in any EST. Translation of the DNA sequence gave riseto a 299 amino acid sequence (FIG. 13) containing structural motifstypical of cyclase enzymes (Prasad, G S, 1996 Nature Struct. biol.3:957-964). In particular, the SM38 protein contains conserved aminoacid residues that align with critical catalytic and active siteresidues found in the Aplysia cyclase enzyme (Munshi C, et al., 1999, J.Biol. Chem. 274:30770-30777) and in mammalian CD38 (Munshi C, et al.,2000, J. Biol. Chem. 275:21566-21571; Graeff R., 2001, J. Biol. Chem.276:12169-12173) (FIG. 15A-B). Additionally, cysteine residues that arecritical for the assembly of the tertiary structure of the cyclaseenzymes (Prasad G S, et al., 1996, NAture Struct. biol. 3:957-964) arealso conserved in SM38; (FIG. 14A-B). Importantly, the SM38 cDNAsequence encodes for a complete cyclase enzyme domain.

Based on these results, we have shown that Schistosomes such as S.mansoni encode a protein (SM38) that is highly homologous at thestructural level to enzymes that are capable of catalyzing theproduction of the calcium mobilizing second messenger, cADPR. SinceSchistosomes also express RyRs which release intracellular calcium inresponse to cADPR, it is predicted that SM38 will be able to regulatecalcium response in Schistosomes. Furthermore, since regulation ofcalcium influx, particularly in Schistosome muscle fibers can result inparalysis and clearance of the worm, we predict thatagonists/antagonists of the SM38 and RyR pathways in Schistosomes may beeffective as anti-helminth drugs.

9. EXAMPLE Characterization of SM38

The subsection below demonstrates that Schistosoma SM38 is structurallysimilar to all of the other cyclase family members and is able tocatalyze NAD⁺ glycohydrolase, ADP-ribosyl cyclase, cADPR hydrolase andtransglycosidation reactions. The subsection also demonstrates that SM38is expressed as a GPI-anchored protein on the outer tegument of adultworms, and is therefore an ideal vaccine target candidate as well as apotential target for small molecule enzyme antagonists.

9.1. Materials and Methods 9.1.1. SM38 Cloning

A blast search using the consensus amino acid sequence for cyclasefamily members (Prasad, et al., 1996, Nature Struc. Biol., 3:957-964)was performed and an EST isolated from Schistosoma mansoni (Accession #AW017229) was identified. Primers specific for the EST (see FIG. 13)were synthesized (Sigma/Genosys), and the 330 bp cDNA was cloned by PCRamplification from a Lambda ZAP II cDNA library constructed from polyA⁺mRNA isolated from adult worm pairs (47). The sequence of the cDNA clonewas verified and the PCR product was used as a probe to isolate multipleindependent overlapping clones from the cDNA library. A full lengthclone containing the entire coding sequence was identified (SM38-native;Accession # AY826981). The nucleotide and amino acid sequence for the S.mansoni SM38 was used in a BLAST search to identify the S. japonicumSM38 orthologue (Accession # AY222890, ref. Hu et al., 2003, Nat Genet.35:139-147).

9.1.2. SM38 Sequence Comparisons and Structural Modeling

The amino acid sequence of S. mansoni SM38 was aligned with the reportedsequences for members of the cyclase family (Accession numbers:NM_(—)007646 (mouse CD38), NM_(—)013127 (rat CD38), AF272974 (rabbitCD38), NM_(—)001775 (human CD38), AF117714 (canine CD38), NM_(—)175798(bovine CD38), NM_(—)009763 (mouse CD157), NM_(—)030848 (rat CD157),NM_(—)004334 (human CD157), AY222890 (S. japonicum SM38), D38536 (A.kurodai cyclase) and M85206 (A. californica cyclase) using the CLUSTAL Wmultiple sequence alignment program (http://www.ebi.ac.uk/clustalw/)(Higgins et al., 1996, Methods Enzymol 266:383-402). The phylogramanalysis comparing the relatedness of S. mansoni SM38 with all otherknown cyclase family members was performed with an evolutionaryrelationship program (http://www.ebi.ac.uk/clustalw/). The threedimensional structure of SM38 was obtained by homology modeling based onthe crystallographic coordinates of both Aplysia ADP-ribosyl cyclase(PDB entry 1lbe) and human BST1/CD157 (PDB entry 1isf) using Modeller(Marti-Renom, et al., 2000, Annu Rev Biophys Biomol Struct 29: 291-325)and energy minimization using AMBER5.

9.1.3. SM38 Constructs

The primary nucleotide sequence of S. mansoni SM38 (SM38-native) wasoptimized for mammalian codon usage, resynthesized (GENEART, Regensburg,Germany) and then cloned into the mammalian expression vector pcDNA3.1(referred to as SM38-opt). To facilitate immunoprecipitation andimmunofluorescence analysis of SM38-opt, the 5′ leader sequence ofSM38-native (see FIG. 15 for sequence), identified by the SignalP(http://www.cbs.dtu.dk/services/SignalP/) (Bendtsen, et al., 2004, J MolBiol, 340:783-795) and Phobius programs (http://pobius.binf.ku.dk/)(Kall et al., 2004, J. Mol. Biol, 338:1027-1036) was replaced with themammalian CD8α leader sequence and a FLAG tag (ref. (Howard et al.,1993, Science, 262:1056-1059); construct referred to as CD8L/FLAG-SM38).To produce recombinant soluble SM38 in COS-7 cells, the GPI-anchorsequence, identified by the consensus sequence for the ω site (site ofGPI attachment, see FIG. 15 for sequence; ref. Eisenhaber et al., 1998,Protein Eng, 11:1155-1161) was removed from CD8L/FLAG-SM38 (constructreferred to as CD8L/FLAG-SM38ΔGPI). To produce soluble recombinant SM38in yeast, the 5′ leader sequence and the 3′ GPI-anchor sequences wereeliminated from SM38-opt and the remaining core ecto-domain sequence wascloned into the Pichia pastoris expression vector pPICZα A (Invitrogen,construct referred to as solSM38-Y). All constructs were sequenced toensure that the insertions and truncations were present and that noartifacts had been introduced to the rest of the SM38 coding regionduring the cloning process.

9.1.4. Generation and Purification of Polyclonal Antiserum to SM38

C57BL/6J mice were bred and maintained in the Trudeau Institute AnimalBreeding facility. All procedures involving animals were approved by theTrudeau Institute Institutional Animal Care and Use Committee and wereconducted according to the principles outlined by the National ResearchCouncil. Mice were vaccinated on days 0, 28 and 56 using a Helios GeneGun (Bio Rad) with 2.1 μm diameter gold bullets that were coated withthe CD8L/FLAG-SM38 vector (1 μg/bullet) and the adjuvant vector,pBOOST-mIL-4/IL1β(0.25 μg/bullet, Invivogen). Serum was collected fromvaccinated mice between days 10-14, 32-38 and 60-70. The antiserum waspooled and the IgG-containing fraction was enriched using Melon Gel IgGSpin Purification kit (Pierce).

9.1.5. SM38 Expression

To express SM38 in mammalian cells, COS-7 cells were transientlytransfected with 30 μg of SM38-native, CD8L/FLAG-SM38 orCD8L/FLAG-SM38ΔGPI DNA per 10 cm plate using Lipofectamine 2000 (Gibco).At 72 h the conditioned media and/or cells were harvested from theplates and analyzed as described below. To express recombinant solubleSM38 in yeast, Pichia pastoris strain GS115 (Invitrogen) waselectroporated (1.8 kV) with the linearized solSM38-Y construct andplated in medium containing zeocin (200 μg/ml). The selectedtransformants were grown in a shaking incubator in 100 ml BMGY (1% yeastextract, 2% peptone, 100 mM potassium phosphate buffer p 16.0, 1.34%yeast nitrogen base without aminoacids, 4×10⁻⁵% biotin, 1% glycerol)containing 1% Bacto casamino acids (Becton Dickinson) to inhibit theprotease activity (54) at 30° C. until culture reached an OD₆₀₀=20. Thecells were harvested by centrifugation at 1500×g for 5 min andresuspended in 50 ml induction medium BMMY (same composition as BMGYexcept that glycerol is replaced by 0.5% methanol). The induction wasmaintained for 48 h at 30° C. (100% methanol is added to 0.5% after 24h). A high-expressing clone was selected and used for large-scaleproduction.

9.1.5. Purification of Soluble Recombinant SM38

To purify soluble recombinant SM38 from CD8L/FLAG-SM38ΔGPI transfectedCOS-7 cells, the conditioned media was collected 72 h post-transfection,adjusted to a final concentration of 150 mM NaCl and then filteredthrough a 0.45 micron filter. The media was passed over an ANTI-FLAGM2-Agarose Affinity Gel column (Sigma, 0.25 ml final packed bed volume).The column was washed and FLAG-tagged SM38 was eluted with 200 μg/mlFLAG Peptide (Sigma) in 250 fractions. The fractions were pooled andconcentrated using a 10K MWCO filter (Millipore). Protein concentrationwas determined with the Nano Drop ND-1000 Spectrophotometer (Nano DropTechnologies, Wilmington Del.) using BSA as a standard.

Recombinant SM38, secreted as a soluble protein in the supernatant ofmethanol-induced Pichia pastoris, was purified in a single step on a1.2×4-cm Blue Sepharose 6 Fast Flow CL-6B column (Amersham Biosciences).After dialysis against 10 mM potassium phosphate buffer (pH 7.4), themedia was loaded at 2 ml/min and the enzyme was eluted with a linear 0-1M gradient of NaCl in the same buffer. This pseudo-affinitychromatography step is performed to ensure that a correctly foldedenzyme is obtained. The protein concentration was determined by the BCAprotein assay (Pierce) using BSA as a standard.

9.1.6. Silver Stain and Western Blot Analysis

Proteins were separated by electrophoresis under reducing conditions on10% SDS-PAGE gels with a 4% stacking gel. Gels were either transferredto PVDF-Plus (Osmonics) for western blot analysis or were fixed, washedand stained with GelCode SilverSNAP Silver Stain Kit (Pierce). Westernblot membranes were blocked with 5% BSA, incubated with biotinylatedanti-FLAG (Sigma, 0.5 μg/ml) or anti-SM38 antiserum (1:2000 dilution) at4° C. for 1.5 h, washed and then incubated with either strepAvidin-HRP(Southern Biotechnology) or anti-mouse Ig (Jackson Immunoresearch) for1.5 h. Blots were developed using either fast or slow-developingchemiluminescence kits (Amersham).

9.1.7. Immunoflourescence Analysis

COS-7 cells were transiently transfected in Lab Tek slide chambers(Nunc) with CD8L/FLAG-SM38 or an empty vector control. At 72 hpost-transfection, the cells were fixed with 4% paraformaldehyde for 5min and then washed and blocked in dPBS containing 5% BSA. To detectSM38 using the mouse anti-SM38 antiserum, slides were stained withanti-SM38 (1:750 dilution) or normal mouse serum (1:750), washed andthen stained with donkey anti-mouse IgG-Alexa-594 (Molecular Probes). Todetect the FLAG tag of CD8L/FLAG-SM38, slides were incubated withavidin/biotin block (Vector), stained with biotinylated mouse anti-FLAGAb (Sigma), washed and then developed with strepavidin-Alexa-594(Molecular Probes). All slides were counterstained with DAPI mount(Molecular Probes) and viewed with a Zeiss Axioplan 2 microscope(Oberkochen, Germany) under brightfield and fluorescence using a 560/40bandpass filter to view Alexa-594 and 330/80 bandpass filter to viewDAPI. Images were captured at 40× original magnification with a ZeissAxioCam digital camera and were overlayed using the Zeiss proprietarysoftware, Axiovision 3.0.6.0.

9.1.8. Localization of SM38 in S. mansoni Parasites

Cryosections of adult worms were stained with affinity-purified IgGsisolated from anti-SM38 mouse serum (5 μg/ml) or purified normal mouseIgG. After washing, the sections were then stained with biotinylatedsecondary anti-mouse IgG (5 μg/ml; Jackson Immunoresearch) washed andthen stained with the far-red fluorophore, AlexaFluor 647-conjugatedstreptavidin (5 μg/ml; Molecular Probes). Live and acetone-fixedparasites (adult worms and mechanically transformed schistosomules; 3 hold) were whole-mounted and stained with anti-SM38 IgG as described forthe cryosections. Cyrosections and whole mount parasites were examinedusing a Bio-Rad MRC-1024 confocal microscope equipped with Krypton-Argonlaser and 522 nm and 650 nm filters. SM38 was visualized with AlexaFluor647 which emits at a maximum wavelength of 647 nm, whereauto-fluorescence produced by phenolic compounds in schistosome sectionsis not detected. The fluorescent three-dimensional structures of wholemount adult worms were reassembled from the sub-micron laser sectionsusing a voxel-based three-dimensional (3-D) imaging program (Voxx, v. 2,55).

9.1.9. Flourometric Enzyme Assays

NAD⁺ glycohydrolase activity was assayed fluorometrically using1,N⁶-etheno-NAD⁺ (s-NAD⁺, Sigma) as previously described (Muller, etal., 1983, Biochem. J., 212:459-464). Briefly, aliquots of cell lysates,conditioned media, purified SM38 protein (25-200 ng), SM38immunoprecipitated on sepharose beads (25 μl beads) or live S. mansoniworms (10 worms/assay) were suspended in HBSS in 96 well blackmicroplates (Corning) in the presence of 40-400 μM ε-NAD⁺ (100 μl finalvolume) at 37° C. in a SpectraMAX GeminiXS fluorescence plate reader(Molecular Devices, Sunnyvale Calif.). The fluorescence emission at 410nm (excitation at 300 nm) of the fluorescent product s-ADPR was thenfollowed for the next 30 min. Data is represented in relativefluorescence units (RFU) after blanking at time 0. The GDP-ribosylcyclase activity was assayed similarly using NGD⁺ (Sigma, 80-800 μM) asthe substrate (Graeff, et al., 1994, J. Biol. Chem., 269:30260-30267)and following the appearance of the fluorescent product cyclicGDP-ribose (emission 410 nm, excitation 310 nm).

The ADP-ribosyl cyclase activity was measured essentially as previouslydescribed (Graeff, et al., 2002, Biochem J 361:379-384) using a cyclingassay. The enzyme was incubated with NAD⁺ (200 μM) in buffer A (1 mlfinal volume) until the reaction reached 50% completion. The enzyme waseliminated by centrifugation for 10 min at 5000×g on ultrafiltrationunits (Vivaspin 2, Vivascience). The contaminating nucleotides wereremoved (overnight incubation at 37° C. in the presence of nucleotidepyrophosphatase, alkaline phosphatase and NAD⁺ glycohydrolase followedby elimination of the three enzymes on ultrafiltration units) and thequantity of cADPR formed was then estimated using the cycling assay.Briefly, the conversion of the cADPR present in the samples into NAD⁺was performed by incubating the sample 1 h at room temperature with 0.1μg/ml Aplysia californica ADP-ribosyl cyclase and 10 mM nicotinamide in100 mM sodium phosphate buffer, pH 8.0. The cycling reaction was thenallowed to proceed in the same buffer in the presence of 0.8% ethanol,10 mM nicotinamide, 40 μg/ml BSA, 20 μg/ml diaphorase (treated withcharcoal), 20 μg/ml alcohol dehydrogenase, 10 μM FMN and 20 μMresazurin. The increase in resorufin fluorescence (excitation at 544 nmand emission at 590 nm) was measured every minute for 2 h using afluorescence plate reader (FluoStar from BMG Labtechnologies Inc).

9.1.10. Non-Flourometric Enzyme Assays

All three activities of SM38 were measured using recombinant enzymepurified from Pichia pastoris. The NAD⁺ glycohydrolase activity wasmeasured under saturating (420 μM) or limiting (25 μM) amounts of NAD⁺in the presence of [adenosine-U-¹⁴C]NAD⁺ (2.5×10⁵ dpm) as previouslydescribed (Lund, et al., 1999, J. Immunol., 162:2693-2702). The enzymewas suspended in 10 mM potassium phosphate buffer, pH 7.4 (buffer A) andincubated at 37° C. with substrate (200 μl final volume). At selectedtimes, aliquots (50 μl) were removed and enzyme activity was stopped byadding ice-cold perchloric acid (2% final concentration). Afterneutralization with 3.5 M K₂CO₃, precipitated proteins were removed bycentrifugation. Product formation was then monitored by HPLC asdescribed below. The same experimental conditions were used with NGD⁺ toassay both NGD⁺ glycohydrolase and GDP-ribosyl cyclase activities.Product formation was then monitored by HPLC on a C₁₈ column asdescribed below.

The cADPR hydrolase activity was measured by incubating cADPR (20 μM) at37° C. in buffer A in the presence of enzyme (200 μl reaction volume).Aliquots were removed at different time points, treated as describedabove and analyzed by HPLC on a C₁₈ column.

The transglycosidation of NADP⁺ was measured by incubating recombinantsoluble SM38 with 1 mM NADP⁺ and 20 to 40 mM nicotinic acid at 37° C.for 20 min in a 10 mM potassium phosphate buffer, pH 6.0 or 7.4 (finalvolume 500 μl). The reaction mixture was analyzed by HPLC on anion-exchange column (see below). HPLC analysis of the reaction productswere performed on aliquots on a C₁₈ column (see below).

9.1.11. Analysis Of Reaction Products by HPLC

Product analysis was performed by HPLC on 300×3.9 mm μBondapack C₁₈column (Waters Assoc., Milford Mass.). The compounds were elutedisocratically at a flow rate of 1 ml/min with a 10 mM ammonium phosphatebuffer, pH 5.5, containing 0.8-1.2% (v/v) acetonitrile and detected byradiodetection (Flo-one, Packard Radiometric Instruments, Meriden Conn.)when using [¹⁴C]NAD⁺ or by absorbance recordings at 260 nm. Theformation of NAADP⁺ was assessed by analysis on 150×4.6 mm AG MP-1column (Interchrom) using a Shimadzu HPLC system. The elution wasperformed at a flow rate of 1 ml/min with a non-linear gradient startingwith 100% solvent A (water). The percentage of solvent B (1.5% TFA) waslinearly increased to 1% in 2 min, 2% in 4 min, 4% in 9 min, 8% in 13min, 16% in 17 min, 32% in 21 min, 100% in 25 min and maintained at 100%for 10 min. Peaks were identified by comparison with authentic samplesand areas obtained from UV recordings were normalized using the molarextinction coefficients of the reaction products.

9.1.12. Enzyme Kinetics

The assays were conducted at 37° C. in buffer A in the presence ofsoluble recombinant enzyme using eight different substrateconcentrations. Reaction progress was obtained either by HPLC analysisof aliquots removed at given times or, alternatively, in the case ofNGD⁺, by monitoring the change of fluorescence at 410 nm (λ_(exc)=310nm). Kinetic parameters were determined from the plots of the initialrates as a function of substrate concentration, according toMichaelis-Menten kinetics, using a non-linear regression program(GraphPad, Prism).

9.1.13. Preparation of Cell and Worm Lysates and Membrane Microsomes

COS-7 cells were transiently transfected with CD8L/FLAG-SM38,CD8L/FLAG-SM38ΔGPI or empty vector for 72 h. Cells were collected,washed in dPBS and frozen until used. Adult S. mansoni worm pairs werecollected from the portal vein of 45 day infected golden hamsters,washed in dPBS and frozen. COS-7 cell pellets and worm pellets werelysed in 10 mM Tris/HCl (pH 7.3), 0.4 mM EDTA, protease inhibitors and1% Triton X-100 (v/v) and detergent-soluble proteins were collected. Insome experiments, the total lysates were used to analyze SM38 enzymeactivity while in other experiments, SM38 was immunoprecipitated fromthe lysates using either Anti-FLAG sepharose beads or protein G beadscoated with anti-SM38 antiserum.

To prepare membrane fractions from adult S. mansoni worms, 2 g of wormswere disrupted at 4° C. with a potter in 10 mM potassium phosphatebuffer (pH 7.4) containing 1 mM EDTA and 0.1 mM phenylmethylsulfonylfluoride (PMSF) as protease inhibitors. The homogenate was centrifugedat 10,000×g for 15 min and the post-mitochondrial supernatant wascentrifuged at 100,000×g for 60 min to obtain the membrane microsomalfraction. The membrane fraction was resuspended in 2 ml 10 mM potassiumphosphate buffer (pH 7.4).

9.1.14. Phospholipase C Treatment

To cleave GPI-anchored proteins from transiently transfected COS-7cells, the cells were washed with HBSS at 48 h post-transfection andthen resuspended in HBSS in the presence or absence of 0.2 Uphosphatidylinositol-specific phospholipase C (PI-PLC from Bacilluscereus, Sigma) at 37° C. for 2 h. The media was removed and assayed forNAD⁺ glycohydrolase activity using ε-NAD⁺ as a substrate. To cleaveGPI-anchored proteins from S. mansoni membrane microsome fractions, 200μl of the microsomes were incubated in the presence or absence of 1 U ofPI-PLC for 2 h at 30° C. The fractions were then centrifuged at100,000×g for 60 min and the supernatant and microsomes were tested forNAD⁺ glycohydrolase and NGD⁺ cyclase activity as described above. Tocleave GM-anchored proteins from S. mansoni adult worms, 10 live wormswere suspended in 200 μl HBSS in the presence or absence of 0.4 U PI-PLCat 37° C. for 2 h. The media was collected and assayed for NAD⁺glycohydrolase activity, GDP-ribosyl cyclase activity andpyrophosphatase activity as described above. Alternatively, the mediawas electrophoresed on an SDS-PAGE gel and analyzed by silver stainingor western blot with the anti-SM38 Ab as described above.

9.1.15. Endoglycosidase Treatment

CD8L/FLAG-SM38 (70 ng) was incubated in 50 mM NaH₂PO₄ (pH 5.5) in thepresence or absence of 0.05 U Endoglycosidase F1 (Sigma) for 1.5 h. Thetreated and untreated protein samples were then analyzed by SDS-PAGE andsilver staining.

9.1.16. SM38 mRNA Transcription Analysis

Analysis of SM38 mRNA levels in different developmental stages wasperformed by semi-quantitative RT-PCR as previously described (Osman etal., 2004, J. Biol. Chem., 269:6474-6486). Briefly, total RNA wasextracted from different developmental stages, representing growth inboth mammalian and molluscan hosts, using TRIzol reagent (Invitrogen).All RNA samples were reverse transcribed using a random decamer, andSuperScript Reverse Transcriptase II (SSRTaseII; Invitrogen) followingvendor's recommended conditions. Complementary DNA (cDNA) samples werethen used as templates in PCR reactions using specific primers for SM38(fwd primer corresponding to by 536-562; rev primer complementarysequence of by 836-862 of the SM38 cDNA, yielding 327 by PCR product).PCR reactions were separated by electrophoresis in 2% agarose gels,ethidium bromide-stained and analyzed using gel-documentation system(GelDoc 1000; Bio-Rad) and quantified using Quantity One software(version 4.2.3; Bio-Rad). A negative control reaction consisting ofreverse transcription reaction mix of adult worm pair total RNA butlacking SSRTaseII (—RT control) was also included. Specific primers forS. mansoni α-tublin gene (GenBank Accession #: M80214; by 424-444 andthe complementary sequence of by 777-801 as forward and reverse primers,respectively, yielding 378 by PCR product) were used to amplify a PCRproduct that served as a constitutively transcribed control and was usedto adjust the input amounts of cDNA templates in PCR reactions ofdifferent developmental stages. In order to ensure that theamplification products were analyzed in the exponential phase and belowsaturation limits (PCR plateau), the number of PCR cycles was alsovaried. Twenty-four cycles were used for α-tubulin while 26 cycles wereused to amplify SM38 PCR products. All variables were considered andcompensated for in data analysis.

9.2. Results 9.2.1. Platyhelminthes Express a Novel Member of theADP-Ribosyl Cyclase Family

The mammalian cyclase, CD38, plays an important functional role inregulating Ca²⁺ signaling in a variety of cell types (Lee, H. C., 2004,Curr. Mol. Med., 4:227-237). To identify other members of the cyclasefamily, the published consensus sequence for ADP-ribosyl cyclases(Prasad, et al., 1996, Nature Struc. Biol., 3:957-964) was used tosearch the public DNA and protein data-bases. A single 330 by EST(Accession # AW017229) that was 27% similar at the amino acid level tothe consensus cyclase sequence (Prasad, et al., 1996, Nature Struc.Biol., 3:957-964) was identified. The EST identified was isolated fromSchistosoma mansoni, a member of the phylum Platyhelminthes. To obtainthe complete cDNA, PCR primers within the EST were designed and wereused to amplify the sequence from a S. mansoni cDNA library. The PCRfragment was then used to probe the S. mansoni cDNA library and identifya full-length clone of 1034 bp containing a 303 amino acid open readingframe giving rise to a protein with a predicted molecular weight of 36kDa (FIG. 13A). As shown in FIG. 13B, the open reading frame for thenovel S. mansoni gene encoded a protein that is 21% identical to thehuman cyclases CD38 and CD157 (37% and 38% similarity respectively), and24% identical to the A. californica cyclase (39% similarity). Thecritical cysteine residues necessary for the 5 conserved intra-disulfidebonds found in most cyclases (Prasad, et al., 1996, Nature Struc. Biol.,3:957-964) are present in the novel S. mansoni sequence (FIG. 13B).Likewise, the crucial catalytic glutamate (E202) found deep within thenicotinamide binding pocket of all known cyclases (Munshi et al., 2000,J. Biol. Chem. 275:21566-21571) as well as a substrate-bindingtryptophan residue (W165) that lines the rim of the nicotinamide-bindingpocket of cyclases (Munshi et al., 2000, J. Biol. Chem. 275:21566-21571)are both conserved in the novel S. mansoni protein (FIG. 13B). Finally,the novel S. mansoni protein has a high degree of similarity (47%) tothe other cyclase family members within the “TLED signature domain”(Munshi et al., 2000, J. Biol. Chem. 275:21566-21571) that containsresidues which localize to the active site pocket (FIG. 13B).

The nucleotide and amino acid sequence from SM38 was then used to searchthe public data-bases to identify any potential SM38 orthologues. Duringthe time between our two data-base searches, a transcriptosome analysisfor the related trematode, Schistosoma japonicum, was published and aclone with significant homology to SM38 and the mammalian cyclase CD38was reported (Accession # AY222890, ref. Hu, et al., 2003, Nat Genet,35:139-147). In fact, the degree of identity between the amino acidsequence of S. mansoni SM38 and S. japonicum SM38 was greater than 56%and the sequences were 74% similar (FIG. 15), suggesting that thisprotein likely represents the S. japonicum SM38 orthologue. Next, twoSM38 sequences were compared to all of the other cyclase family membersin order to generate a phylogram. As shown in FIG. 13A, SM38 is mostclosely related to the Aplysia cyclases and then to the mammaliancyclase CD157. Since the three dimensional structures for both theAplysia cyclase and CD157 have been previously published (Prasad, etal., 1996, Nature Struc. Biol., 3:957-964, Yamamoto-Katayama, et al.,2002, J. Mol. Biol., 316:711-723), these structures were used to model,by homology, the structure of SM38 and its putative active site. Asshown in FIG. 13B, the modeled structure for SM38 was very similar tothat of CD157, particularly within substrate-binding groove. The modelalso demonstrates that the highly conserved residues found within theactive site of all of the known cyclases, including the catalyticGlu²⁰²), the Glu¹²⁴ of the “signature domain” that regulates hydrolaseactivity (Graeff, et al., 2001, J Biol. Chem. 276:12169-12173), and theTrp¹⁶⁵ that influences substrate positioning (Munshi et al., 2000, J.Biol. Chem. 275:21566-21571) are localized within the active site pocketof SM38 (FIG. 13C). At odds with the other cyclases, S. mansoni SM38 hasa histidine residue at position 103, while the equivalent position inall other known cyclases is invariably occupied by tryptophan, a residuethat plays a role in substrate binding within the active site (Munshi etal., 2000, J. Biol. Chem. 275:21566-21571). Interestingly, the histidineresidue in S. mansoni SM38 is also located within the active site grooveof our model (FIG. 13C) and is conserved in S. japonicum SM38 (FIG. 15).Taken together, the data indicate that SM38 is found in at least twomembers of the Platyhelminthes phylum, that the amino acid sequence ofSM38 shares a significant degree of homology with other cyclase familymembers and that the predicted structure of the SM38 protein isstrikingly similar to the other cyclase family members.

9.2.2. Recombinant SM38 is a GPI-Anchored Membrane NAD⁺ Glycohydrolase

To determine whether SM38 is a functional enzyme, a heterologous(mammalian) in vitro expression system for SM38 was developed. Sincepreferred codon usage between Platyhelminthes and mammals is quitedifferent, the S. mansoni SM38 cDNA was re-synthesized to facilitatetranslation in mammalian cells (SM38-opt). SM38-opt was then transientlyexpressed in COS-7 cells and the NAD⁺ glycohydrolase (NADase) activityin the supernatant or cell lysates was determined by measuring thehydrolysis of 1,N⁶-etheno-NAD⁺ (ε-NAD⁺). Hydrolysis of ε-NAD⁺ by cyclasefamily members results in the generation of fluorescent ε-ADPR that caneasily be detected using a spectrofluorimeter (Muller et al., 1983,Biochem. J., 212:459-464). NADase activity was measured in thesupernatant of the transfected cells since analysis of the SM38 sequenceusing different protein structure prediction programs (Bendtsen, et al.,2004, J. Mol. Biol., 340:783-795; Kall et al., 2004, J. Mol. Biol.,338:1027-1036) indicated that SM38 had a signal sequence (FIG. 15) butno obvious transmembrane domain and would therefore be secreted.However, NADase activity in the transfected cell supernatants was notdetected (FIG. 19A). to determine whether SM38 was secreted, transientlytransfected cells were lysed in 1% Triton X-100 and then NADase activityin the cell lysate was measured. Interestingly, abundant NADase activityin the lysate of COS-7 cells transfected with SM38-opt was detected(FIG. 19A), but no activity in cells transfected with the emptyexpression vector was observed (data not shown). These results stronglyindicate that SM38 is expressed as either a transmembrane or cytosolicprotein.

To determine the subcellular localization of SM38, it was necessary todirectly visualize SM38 within cells. To facilitate this, the 5′ signalsequence of SM38-opt was replaced with a mammalian signal sequence (CD8αleader) followed by a FLAG tag (CD8/FLAG-SM38). To assess whether theFLAG-tagged recombinant protein could be detected and purified withanti-FLAG reagents, COS-7 cells were transiently transfected with theCD8L/FLAG-SM38 construct. The cells were lysed in detergent and SM38 waspurified over an anti-FLAG affinity column. The FLAG-tagged SM38 waseluted, the purified protein was separated on SDS-PAGE, and then awestern blot was performed using an anti-FLAG antibody. A protein ofapproximately 48 kD was detected in transfected cell lysates (FIG. 19B).Next, to determine where SM38 was localized in the transfected cells,COS-7 cells were transiently transfected with the CD8L/FLAG-SM38construct and then a biotinylated anti-FLAG antibody was used to performimmunofluorescence analysis. As shown in FIG. 2C, the plasma membrane ofCOS-7 cells that were transfected with CD8L/FLAG-SM38 specificallystained with the anti-FLAG antibody, while no staining was observed incells that were transfected with an empty expression vector (FIG. 19D).Therefore, these data indicate that SM38, at least when expressed inmammalian cells, is membrane-associated.

SM38 was not predicted to have a transmembrane domain by any of thecommonly used protein structure prediction programs. However, CD157, theclosest mammalian relative of SM38, is a membrane-associatedGPI-anchored protein. Therefore, the 3′ amino acid sequence of SM38 wasre-examined to see if a GPI anchor motif could be identified. Althoughthe SM38 3′ sequence did not conform with most of the motifs that havebeen described for mammalian GPI anchors (Eisenhaber, et al., 2003Bioessays 25; 367-385), a potential GPI-anchor site was identified(ω-site, see FIG. 15) with the help of an algorithm written byEisenhaber et. al. (Eisenhaber, et al., 1998 Protein Eng. 11;1155-1161). To determine whether SM38 is GPI anchored, it was testedwhether SM38 can be cleaved from the membrane of CD8L/FLAG-SM38transfected COS-7 cells by Phosphotidyl inositol-specific PhospholipaseC (PI-PLC), a GPI-anchor lipase. The media from transfected andnon-transfected COS-7 cells was removed, the cells were washed, and thenthe cells were incubated in the presence or absence of PI-PLC. Thesupernatant was collected 2 hours later and NADase activity was measuredusing ε-NAD⁺ as a substrate. As expected, NADase activity was notdetected in the supernatant of cells transfected with the empty vector,regardless of whether PI-PLC was added (FIG. 19E). Likewise, no activityin the supernatant of cells transfected with CD8L/FLAG-SM38 andincubated in media alone was detected (FIG. 19E). In contrast, abundantNADase activity was observed in the supernatant collected from theCD8L/FLAG-SM38 transfected cells that were incubated with PI-PLC (FIG.19E). Identical results were observed when the experiment was performedusing COS-7 cells transfected with the original native form of SM38.Thus, SM38, like CD157, is expressed as a GPI-anchoredmembrane-associated protein in mammalian cells.

9.2.3. SM38 is a Multifunctional Enzyme

Cyclases are multi-functional enzymes that catalyze several reactionsincluding the hydrolysis of NAD⁺ to produce ADPR (NAD⁺ glycohydrolaseactivity), the cyclization of NAD⁺ into cADPR (ADP-ribosyl cyclaseactivity), the hydrolytic conversion of cADPR into ADPR (cADPR hydrolaseactivity) and the transglycosidation of NADP⁺ (Eisenhaber, et al., 2003Bioessays 25; 367-385). To better characterize the enzymatic propertiesof SM38, a secreted soluble form of the SM38 ecto-domain by deleting theGPI-anchor motif was produced (see FIG. 15) in a FLAG-tagged SM38construct (CD8L/FLAG-SM38ΔGPI). COS-7 cells were transiently transfectedwith the new construct, and the supernatant was collected after 72 h.NADase activity in the supernatant and in the cell lysate was measuredusing e-NAD⁺ as a substrate. As expected, the enzyme activity was nowhighly enriched in the supernatant fraction and was found at only verylow levels in the cell lysates (FIG. 20A). Next, the secreted SM38protein was purified from the supernatant on an anti-FLAG column and, asexpected, affinity purification of SM38 resulted in significantenrichment of the NADase activity (FIG. 20A, inset). The affinitypurified soluble SM38 was then analyzed by SDS-PAGE and silver-stainingand two protein species of 43 and 46 kD were observed (FIG. 20B). Toensure that both of the eluted proteins were bona fide SM38, westernblot analysis using an anti-FLAG antibody was performed. As shown inFIG. 20B, both protein species were recognized by the anti-FLAGantibody.

Since two protein forms for SM38 were identified and both forms wereconsiderably larger than the predicted molecular weight of soluble SM38(˜30 kDa) the possibility that SM38 might be glycosylated wasconsidered, particularly since the SM38 sequence contained 4 potentialN-linked glycosylation sites (FIG. 15). Therefore, the purifiedrecombinant SM38 was treated with Endoglycosidase F1 (Endo-F) to cleaveN-linked sugars and the molecular weight of the treated proteins wasthen determined. Treatment of SM38 with Endo-F reduced the size of bothforms of SM38 by about 2 kD to 44 and 41 kD (FIG. 20B). Thus, solublerecombinant SM38 is expressed in two isoforms, both of which areglycosylated.

Although SM38-specific NADase activity was easily detected using ε-NAD⁺as a substrate and measuring product formation fluorimetrically (i.e.,FIG. 20A), larger quantities of soluble SM38 were needed to bettercharacterize the catalytic properties of SM38. Therefore, the productionand purification of soluble SM38 was scaled up using a Pichia pastorisexpression system and an affinity gel purification scheme routinelyemployed to purify other cyclases and NAD⁺ glycohydrolases(Cakir-Keifer, et al., 2000 Biochem. J. 349; 203-210). Similar to whatwas observed using the mammalian expression system (FIG. 20B), it wasfound that soluble SM38 is expressed in Pichia in two isoforms of 43 and44 kD (FIG. 20C). The NAD⁺ glycohydrolase activity of the purified SM38was then determined by incubating the recombinant enzyme with saturatingamounts of radio-labeled NAD⁺ and analyzing product formation by HPLC(FIG. 20D). As indicated in Table II, the specific activity of SM38 wascalculated to be 13.2 μmol/min/mg of SM38 protein; a value that is inthe same range as that reported for mammalian cyclases such as CD38(Howard, et al., 1993 Science 262; 1056-1059; Cakir-Keifer, et al., 2000Biochem. J. 349; 203-210).

TABLE II Kinetic parameters for the transformation of substrates bySM38^(a) K_(m) V_(max) k_(cat) k_(cat)/K_(m) Substrate μM μmol/min/mgs⁻¹ M⁻¹s⁻¹ NAD⁺ 38.5 ± 6.8 13.2 ± 2.6 6.51 ± 1.28 1.69 × 10⁵ NGD⁺ 23.4 ±1.1  4.8 ± 0.1 2.37 ± 0.05 1.01 × 10⁵ NADP⁺ 13.7 ± 2.9 39.2 ± 1.6 19.34± 0.79  14.12 × 10⁵ 

*All reactions were performed at 37° C. in 10 mM potassium phosphatebuffer (pH 7.4) in the presence of purified recombinant SM38 produced inPichia pastoris.

Interestingly, using HPLC to measure product formation, ADPR was easilyvisualized but measurable cADPR was not detected (FIG. 21D), despitevarying the pH of the assay between 5.0-8.0 (data not shown). Since theamount of cADPR produced by SM38 was less than the limit of detection byHPLC, (i.e. <1% of the radio-labeled reaction products), a verysensitive cycling assay (Graeff, et al., 2002 Biochem. J. 361; 379-38)was next used to measure cADPR production by recombinant soluble SM38.Although cADPR was detect rf by this method, the amount produced understeady-state conditions was very small and represented only 0.016% ofthe total reaction product ADPR.

These data indicate that SM38 is either a much more efficient NAD⁺glycohydrolase than ADP-ribosyl cyclase or that the produced cADPR israpidly hydrolyzed by SM38 to ADPR (cADPR hydrolase activity). Todistinguish between these possibilities, the cADPR hydrolase activity ofSM38 was measured. It was found that SM38 is a very poor cADPR hydrolasewith an activity of less than 6 nmol/min/mg protein, which is 3 ordersof magnitude less than the NAD⁺ glycohydrolase activity of SM38 (TableII). Therefore, it seemed unlikely that SM38 was degrading the cADPR asrapidly as it produced it and more likely that SM38 produces very lithecADPR under steady-state conditions. To specifically test whether SM38is competent to catalyze a cyclase reaction, the purified enzyme wasincubated with NGD⁺, an analogue of NAD⁺, which is efficiently convertedto cyclic GDP-ribose (cGDPR) by many of the mammalian cyclase familymembers and is easily detected fluorometrically (Graeff, et al., 1994 J.Biol. Chem. 269; 30260-30267). NGD⁺ proved to be an excellent substratefor SM38 (K_(m)=23.4 μM, Table II) and as shown in FIG. 20E was quiteefficiently cyclized. The cyclization/hydrolysis ratio was approximately6.0 (FIG. 20E), and the specific activity under steady state conditionswas 4.8 μmol/min/mg protein (FIG. 20F, Table II); all of which are quitecomparable to that reported for mammalian cyclases such as CD38(Berthelier, et al., 1998 Biochem J. 330; 1383-1390). Thus, these datashow that, while SM38 is a very inefficient ADP-ribosyl cyclase, thisenzyme is mechanistically competent to catalyze the production of cycliccompounds such as cGDPR.

Next, whether SM38 was able to use NADP⁺ as substrate and to catalyze atransglycosidation reaction in the presence of nicotinic acid wastested. As indicated in Table II, NADP⁺ proved to be an excellentsubstrate of SM38 and, in terms of catalytic efficiency (k_(cat)/K_(m)),NADP⁺ was an even better substrate than NAD⁺ (8-fold better, see TableII). This is contrast with human CD38 for which NAD⁺ was the favoredsubstrate (Berthelier, et al., 1998 Biochem J. 330; 1383-1390). As shownby HPLC (FIG. 20G), the pyridine-base exchange reaction was readilycatalyzed by SM38, at pH 6.0, indicating that this enzyme, like CD38 andthe Aplysia cyclase, is able to synthesize the Ca²⁺-mobilizing messengerNAADP⁺. As noted before for the other members of the cyclase family, theformation of NAADP⁺ was less efficient under neutral pH conditionscompared to more acidic pH conditions (3-fold reduction in NAADP⁺formation at pH 7.4, compared to pH 6.0, data not shown).

In addition to catalyzing a base-exchange reaction using NADP⁺ as thesubstrate, it was also found that SM38 can catalyze a base-exchangereaction using NAD⁺ as the substrate in the presence of isoniazid (INH,not shown). This pyridine has been widely used to classify the mammalianNAD⁺ glycohydrolases (Table III) into either ‘INH-sensitive’ (e.g.bovine CD38) or ‘INH-insensitive’ (e.g. human CD38) NADases (Zatman, etal., 1954 J. Biol. Chem. 209; 453-466) and SM38 appears to be a memberof the latter category of NADases.

Finally, like the other members of the cyclase family, SM38 can catalyzethe methanolysis of NAD⁺ leading to the formation of β-methyl ADP-ribose(Table III). On a molar basis, methanol was found to react about 5-foldfaster than water (H. M-S. and F. S. unpublished). Moreover, thepresence of 1-3 M methanol did not affect the overall turnover rate ofthe NAD⁺ solvolysis reactions (hydrolysis and methanolysis). These dataare consistent with the cleavage of the nicotinamide-ribose bond beingthe rate-limiting step of the kinetic mechanism of SM38, again similarto what has been previously shown for other cyclase family members suchas CD38 (Schuber, et al., 2004 Curr. Mol. Med. 4; 249-261; Cakir-Kiefer,et al., 2001 Biochem J 358; 399-406; Muller-Steffner, et al., 1994Bioch-Biophys. Res. Commun. 204; 1279-1285). Taken altogether, the dataindicate that the enzymatic properties of SM38 are similar to othermembers of the cyclase family, and that SM38, can catalyze theproduction of ADPR, NAADP⁺, and to a lesser extent, cADPR.

TABLE III Comparison of SM38 enzyme properties with cyclase familymembers Mammalian Properties SM38 cyclases Aplysia cyclase Hydrolysis ofNAD⁺ yes yes very low Formation of cADPR very low low yes Hydrolysis ofcADPR very low yes very low Formation of cGDPR yes yes yesTransglycosidation yes yes yes Sensivity to INH no yes/no NDMethanolysis of NAD⁺ yes yes very low Inhibition by araF-NAD⁺ weak Ki nMrange weak (IC₅₀ > 10 μM) (IC₅₀ > 10 μM)

9.2.4. SM38 Expression is Developmentally Regulated in S. mansoni

To determine when and where SM38 is expressed during the life-cycle ofS. mansoni, a semi-quantitative PCR approach with SM38-specific and S.mansoni α-tubulin specific primers was used to determine when SM38 mRNAtranscripts are expressed during schistosome development. SM38transcripts were not detectable in S. mansoni eggs or in uninfectedBiomphalaria glabrata snails (intermediate hosts) but were easilyobserved in the S. mansoni-infected snails (FIG. 21A). SM38 expressionthen declined to undetectable levels in the S. mansoni cercariae,schistosomules and in immature day 21 worm pairs (FIG. 21A-B). Incontrast, SM38 expression was dramatically increased in day 28 and day35 worm pairs, coinciding with male-female worm pairing, and was thenmaintained in both male and female mature adult worm pairs (FIG. 21A-B).Together, these data indicate that SM38 plays a developmentallyregulated signaling function in S. mansoni in both intermediate anddefinitive hosts.

9.2.5. SM38 is Expressed as a Constitutively Active Ecto-Enzyme in theTegument Membrane of Adult S. mansoni

To evaluate whether SM38 protein is expressed and functional in adult S.mansoni worms membrane and cytosolic fractions from homogenized adultworms was purified and NAD⁺ glycohydrolase activity was assessed usingradio-labeled NAD⁺ as a substrate. Identical to our results using theheterologous mammalian expression system, NAD⁺ glycohydrolase activitywas observed exclusively in the membrane microsome fraction of the adultS. mansoni worms. In addition, despite using radio-labeled NAD⁺ as thesubstrate, only the formation of ADPR was detected, while cADPR was notdetected (FIG. 22A), indicating that the native enzyme, like therecombinant version, is a very inefficient ADP-ribosyl cyclase. To testwhether the native SM38 could catalyze a cyclase reaction, membranemicrosomes were incubated with NGD⁺ and the cGDPR accumulation wasmeasured fluorimetrically and by HPLC. As expected, the S. mansonimembrane fraction very efficiently catalyzed a cyclase reaction with acGDPR/GDPR ratio of 12 (FIG. 22B). Next, to determine whether themembrane-associated NAD⁺ glycohydrolase/NGD⁺ cyclase expressed by adultS. mansoni worms was GPI-anchored, the microsomal fraction was incubatedin the presence and absence of PI-PLC and then NAD⁺ glycohydrolaseactivity released into the supernatant was measured. As expected, theSchistosome NAD⁺ glycohydrolase was sensitive to PI-PLC (FIG. 22C).Together, these data indicate that the Schistosoma NAD⁺ glycohydrolaseis a membrane-associated GPI-anchored protein.

Schistosomes, like all flatworms, have an outer membrane, referred to asthe tegument (66). The tegument is composed of a syncytium having aheptalaminar apical membrane and a basal membrane separated by 9 nM ofcytoplasm connecting via a cytoplasmic bridge to subtegumental cytons.To assess whether the Schistosoma NAD⁺ glycohydrolase is associated withthe outer tegument membrane of the adult worm, live Schistosomaparasites were directly incubated with the membrane impermeant substrateε-NAD⁺ and accumulation of fluorescent ε-ADPR was measured. As shown inFIG. 22D, as few as 10 live S. mansoni adult worms were able toefficiently catalyze the NAD⁺ glycohydrolase reactions, indicating thatadult S. mansoni parasites express an enzyme(s) with the properties ofSM38 on the outer tegument. To determine whether the NADase associatedwith the outer tegument of S. mansoni adult worms is GPI-anchored, 10live parasites were incubated in the presence or absence of PI-PLC, thesupernatant was collected after two hours and NAD⁺ glycohydrolaseactivity was measured using ε-NAD⁺ as the substrate (FIG. 22E). Culturemedia from the worms that were incubated without PI-PLC was devoid ofNADase activity, however NADase activity in the medium from the wormsthat were treated with PI-PLC was easily detected (FIG. 22E). Finally,to ensure that an authentic NAD⁺ glycohydrolase on the outer tegumentwas being and not a pyrophosphatase (which can also utilize NAD⁺ as asubstrate), the PI-PLC treated supernatant was incubated with1,N⁶-etheno-PyAD⁺, a substrate that is transformed by nucleotidepyrophosphatases into the fluorescent 1,N⁶-etheno-AMP, but cannot beutilized by members of the cyclase family (Muller, et al., 1984 BiochemJ 223; 715-721). No pyrophosphatase activity was detected (data notshown), thus the enzyme present on the outer tegument of adult worms isan authentic GPI-anchored NAD⁺ glycohydrolase.

The data indicate that SM38 or a SM38-like enzyme is expressed as anouter tegument protein by adult S. mansoni worms. In order to directlytest this hypothesis, SM38 specific antibodies were generated byimmunizing and boosting mice with the CD8LFLAG-SM38ΔGPI expressionconstruct. Serum was collected from the immunized mice and thespecificity of the antiserum was assessed by western blot,immunofluorescence and immunoprecipitation. As shown in FIG. 23A, theanti-serum raised in response to the SM38 cDNA vaccine specificallyrecognized soluble recombinant SM38 protein by western blot but did notreact with other purified proteins including ovalbumin and BSA (notshown). Next, to determine whether the anti-SM38 antiserum wouldspecifically recognize membrane-associated SM38 protein, COS-7 cellswere transiently transfected with CD8L/FLAG-SM38 or the empty expressionvector and then the cells were stained with the anti-SM38 antiserumfollowed by a fluorochrome-conjugated anti-mouse immunoglobulinantibody. As expected, the anti-SM38 antiserum did not stain the emptyvector-transfected cells (FIG. 23B) nor did normal mouse serum stainSM38-transfected cells (FIG. 23C). However, identical to what wasobserved when the SM38 transfected cells were stained with anti-FLAGantibody (see FIG. 19C), the anti-SM38 antiserum stained the plasmamembrane of COS-7 cells transfected with CD8L/FLAG-SM38 (FIG. 23D).

To demonstrate that the anti-SM38 antiserum could be used toimmunoprecipitate enzymatically active SM38 protein, COS-7 cells weretransiently transfected with CD8L/FLAG-SM38 or the empty expressionvector, the transfected cells were lysed in Triton X-100 and thedetergent soluble proteins were collected. Immunoprecipitations wereperformed using the anti-SM38 antiserum or normal mouse serum coupled toprotein G beads and then whether the immunoprecipitated proteins wereable to catalyze the hydrolysis of ε-NAD⁺ was analyzed. No enzymeactivity was observed in immunoprecipitates from control transfectedcells regardless of whether normal mouse serum or anti-SM38 antiserumwas used as the immunoprecipitating antibody (FIG. 23E). Likewise, noenzyme activity was detected in the immunoprecipitates using the normalmouse serum and lysates from the SM38-transfected cells (FIG. 23E).However, abundant NAD⁺ hydrolase activity was detected in theimmunoprecipitated proteins isolated from the SM38-transfected cellsusing the anti-SM38 antiserum (FIG. 23E). Together, these data indicatethat the antiserum raised against the SM38 cDNA is specific for SM38 andrecognizes enzymatically active membrane-anchored SM38 as well asdenatured SM38.

To determine whether enzymatically active SM38 protein is expressed byadult S. mansoni worms, adult worms were lysed in Triton X-100, theproteins were collected and immunoprecipitations were performed usingour anti-SM38 antiserum or normal mouse serum as a control. Whether theproteins precipitated from the adult worms were able to hydrolyze ε-NAD⁺was then determined. As shown in FIG. 23F, the anti-SM38immunoprecipitated proteins catalyzed a NAD⁺ glycohydrolase reactionwhile the proteins that were immunoprecipitated with normal mouse serumhad no detectable enzyme activity. Finally, to visualize the native SM38protein expressed by adult S. mansoni worms, live worms were incubatedin HBSS in the presence of PI-PLC. The proteins released into theculture media were collected and concentrated and then the proteins wereanalyzed by silver staining and western blot. As shown in FIG. 23G, alarge number of proteins were either secreted or shed by thePI-PLC-treated adult worms. However, upon western blot analysis usingour anti-SM38 antiserum, a single protein of approximately 43 kDa wasdetected (FIG. 230). Interestingly, the native SM38 protein expressed bythe adult S. mansoni worms was very similar in size to the solublerecombinant SM38 secreted by transfected COS-7 cells (FIG. 23G). NativeSM38 was also detected a two similar-sized protein forms using anti-SM38antibodies to probe western blots of extracts isolated from live wormstreated with NP-40 to enrich for surface-associated proteins (FIG. 16).Taken together these data show that an antiserum raised against SM38, anovel protein encoded by a cDNA isolated from S. mansoni which showssignificant homology to the NAD⁺ glycohydrolase/ADP ribosyl cyclasefamily of enzymes, specifically recognizes a surface-associatedGPI-anchored protein in adult S. mansoni worms that is capable ofcatalyzing NAD⁺ glycohydrolase and NGD⁺ cyclase reactions.

RT-PCR data and western blot experiments using anti-SM38 antibodiesstrongly indicated that SM38 is developmentally expressed as a membraneand tegument-associated protein in adult S. mansoni parasites. Toconfirm these findings, the IgG fraction of the anti-SM38 antiserum wasused to detect the native SM38 protein in adult worm cryosections, wholemount worms and mechanically transformed schistosomules (3 h-old). SM38protein was not detected in either live or acetone-fixed 3 hschistosomules (data not shown). Similarly, no specific fluorescence wasobserved in adult worm cryosections (FIG. 24C) or whole worms (FIG. 24G)when probed with pre-immune normal mouse IgG. In contrast, strongsurface staining along the male gynecophoric canal (arrow; FIG. 24F) aswell as fluorescence of the epithelial tissues surrounding the gut (notshown) could be seen in adult worm cryosections probed with anti-SM38antibody. In addition, surface fluorescence was also detected inacetone-fixed (FIG. 24H) or live (FIG. 24I) whole worms probed withanti-SM38 antibodies. Specific staining was observed on the surface(tubercles, FIG. 24H), male gynecophoric canal (FIG. 24H) and oral andventral suckers of male and female worms (FIG. 24H-I). Thus, these dataindicate that in adult schistosomes SM38 is localized to the outersurface of the parasite. The implications of these results for potentialSM38 vaccination and small molecule antagonist studies is discussed.

10. EXAMPLE CD38 Deficient T Cells do not Induce Lung Inflammation afterAllergen Challenge

The subsection below demonstrates that CD38 expression onallergen-specific T cells (or autoimmune T cells) is required for eithertheir maturation into differentiated effector cells or for theirmigration to sites of inflammation. The subsection also demonstratesthat inflammatory responses in the lungs of allergen-challenged mice isdependent on CD38-expressing T cells.

10.1. Materials and Methods

Naïve T cell receptor (TCR) transgenic T cells specific for ovalbumin(OVA) were purified from the spleens and lymph nodes of normaltransgenic mice (WT T cells) and CD38 deficient transgenic mice (KO Tcells). Either KO or WT OVA-specific T cells (CD45.2⁺) were theninjected into congenic normal hosts (CD45.1⁺) and the host animals werethen sensitized with 10 μg NP-OVA (n=7 mice/group) or PBS administered(n=3 mice/group) intranasally once/day for the next 7 days. On day 8,the cells from the draining lymph node and the lung airways (Bronchialalveolar lavage, BAL) were collected, counted and analyzed by FACS.

10.2. Results

The number of donor OVA-specific T cells with an activated phenotype)(CD45.2⁺ CD4⁺ CD62L^(lo)) present in the lymph node and BAL of the shamand allergen-challenged host is depicted in FIG. 25. The number ofinfiltrating inflammatory cells to the lungs of the mice is indicated inFIG. 26. The CD38 deficient OVA-specific T cells are reduced in numberin both the lymph node and in the lung at the site of inflammation.Similarly, the allergen-induced inflammatory response is suppressed inthe lungs of the mice receiving CD38 deficient T cells. This deficiencyin maturation/migration of T cells which lack CD38 results in a reducedinflammatory response in the lungs of allergen-challenged animals.Therefore, the data suggest that CD38 inhibitors will reduce Tcell-mediated pathology at sites of inflammation such as the lung inasthma as well as inflamed tissues/joints etc. of patients sufferingfrom autoimmune disease.

11. EXAMPLE Priming Of Inflammatory Allergen Specific T Cells is Reducedin CD38 Deficient Mice, Even when the T Cells are from Normal Animals

The subsection below demonstrates that CD38 expression onantigen-presenting cells is required for the expansion ofallergen-specific T cells. The subsection also demonstrates thatexpression of CD38 on antigen-presenting cells promotes cellularinflammation in the airways and lung tissue of allergen challenged mice.

11.1. Materials and Methods

Naïve T cell receptor (TCR) transgenic T cells specific for ovalbumin(OVA) were purified from the spleens and lymph nodes of normaltransgenic mice. The normal OVA-specific T cells (CD45.1⁺) were theninjected into congenic normal hosts (WT, CD45.2⁺) or CD38 deficienthosts (KO, CD45.2⁺). The host animals were then sensitized with 10 μgNP-OVA (n=7 mice/group) or PBS administered (n=3 mice/group)intranasally once/day for the next 7 days. On day 8, the cells from thedraining lymph node and the lung airways (Bronchial alveolar lavage,BAL) were collected, counted and analyzed by FACS.

11.2. Results

The number of donor OVA-specific T cells (CD45.2⁺ CD4⁺) present in thelymph node and BAL of the sham and allergen-challenged host is depictedin FIG. 27. The number of activated CD62Llo donor T cells present in thelymph nodes is also shown in FIG. 27. The number of infiltratinginflammatory cells to the lungs of the mice is indicated in FIG. 28. Arepresentative H&E section of the lungs of OVA challenged WT or CD38 KOmice is also depicted in FIG. 28. Expression of CD38 onantigen-presenting cells is required for the efficient priming,expansion and differentiation of allergen-specific T cells (even whenthe T cells are CD38 sufficient). The reduction in T cell primingobserved in the CD38 deficient mice leads to reduced numbers ofallergen-specific T cells that can induce an inflammatory response inthe lung. Therefore, the data suggest that CD38 inhibitors will reducethe expansion of allergen-specific or autoreactive T cells, resulting inreduced T cell mediated pathology at sites of inflammation such as thelung in asthma as well as inflamed tissues/joints etc. of patientssuffering from autoimmune disease.

12. EXAMPLE Allergen-Induced Inflammatory Responses in the Lungs areReduced in CD38 Deficient Mice

The subsection below demonstrates that CD38 deficient mice are moreresistant to allergen-induced inflammation in the lung.

12.1. Materials and Methods

CD38 deficient (KO) or normal C57BL/6 (WT) mice were primed withovalbumin (OVA) in alum on day 0 (10 ug/mouse administered i.p.) or wereinoculated with PBS. On day 42 post-immunization, animals were eitherleft untreated (prime only) or were challenged with 10 μg OVAadministered intranasally 1 time/day for the next 7 days(prime+challenge group and challenge only group). The lungs wereisolated from all groups of mice one day after the last administrationof OVA and were prepared for histological examination. H&E stainedparaffin sections of a representative animal from each group are shown.

12.2. Results

The inflammatory cell infiltrate is significantly reduced in the lungsof the CD38 deficient mice that were primed and then challenged withOVA. Expression of CD38 on either hematopoietic cells or resident lungcells (epithelial, stromal or fibroblast) is required for the inductionof an inflammatory response in the lungs of mice that have been primedand then sensitized with an allergen administered into the lung airways.Therefore, these data suggest that CD38 inhibitors will block theinduction of allergic responses in patients and/or the chronicinflammation in the lungs of allergic or asthmatic patients.

13. EXAMPLE Diabetes Onset is Delayed in CD38 Deficient Mice 13.1.Materials and Methods

CD38 deficient (KO) or normal BALB/c (WT) mice were injected withStreptozotocin (STZ; 50 mg/kg/mouse) 1 time/day for 5 consecutive days.Blood glucose levels were measured 10 and 17 days after the last STZinjection.

13.2. Results

The multi-low dose STZ treatment resulted in increased blood glucoselevels in both WT and KO animals within 10 days of treatment. However,the hyperglycemia was significantly higher in the WT animals. By day 17,the majority of WT mice were diabetic (7/11 animals with bloodglucose>350 mg/dl) while only a small fraction of the KO mice werediabetic (3/11 animals). CD38 expressing cells facilitate destruction ofthe pancreas in response to STZ treatment indicating that CD38 regulatesimmune-mediated inflammatory responses that cause the destruction of thepancreatic β-cells. Therefore, these data suggest that CD38 inhibitorscould be used to either prevent or delay the onset of autoimmunemediated diabetes.

The present invention is not to be limited in scope by the specificembodiments described herein which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the claims. Variouspublications are cited herein, the contents of which are herebyincorporated, by reference, in their entireties.

1-34. (canceled)
 35. An isolated SM38 polypeptide encoded by a nucleicacid molecule that binds in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate(SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at 68° C. toa nucleotide sequence that encodes the SM38 amino acid sequence of FIG.13A (SEQ ID NO: 5).
 36. The isolated SM38 polypeptide of claim 35comprising the amino acid sequence of FIG. 13A (SEQ ID NO: 5).
 37. Apharmaceutical composition comprising a SM38 polypeptide, or polypeptidefragment thereof, and a pharmaceutically acceptable carrier.
 38. Thepharmaceutical composition of claim 37, further comprising an adjuvant,wherein the SM38 polypeptide, or polypeptide fragment thereof,stimulates an immune response.
 39. An antibody which is capable ofbinding to the SM38 polypeptide of claim
 35. 40. The antibody of claim39 wherein the antibody is a monoclonal antibody.
 41. A method foridentifying a compound that inhibits SM38 enzyme activity comprising (i)contacting SM38 with a test compound and substrate and measuring thelevel of SM38 activity, (ii) in a separate experiment, contacting SM38and substrate and measuring the level of SM38 activity, where theconditions are essentially the same as in part (i) and then (iii)comparing the level of SM38 activity measured in part (i) with the levelof SM38 activity in part (ii), wherein a decrease level of SM38 activityin the presence of the test compound indicates that the test compound isa SM38 inhibitor.
 42. The method of claim 41 wherein the SM38 is apolypeptide expressed in a cell.
 43. The method of claim 41 wherein theSM38 is a purified SM38 polypeptide.
 44. The method of claim 41 whereinthe SM38 enzyme activity is selected from the group of enzyme activitiesconsisting of NAD+glycohydrolase, ADP-ribosyl cyclase andtransglycosidation activity.
 45. The method of claim 41 wherein the SM38enzyme activity is measured by monitoring the rate of formation ofnicotinamide, ADP-ribose (ADPR), cyclic-ADPR (cADPR), or nicotinic acidadenine dinucleotide phosphate (NAADP).
 46. The method of claim 41wherein the SM38 to be contacted in step (i) and (ii) with a testcompound or vehicle control is provided in an array of locations.
 47. Amethod of inhibiting the activity of Schistosoma comprising contactingsaid Schistosoma with an inhibitor of SM38 activity.
 48. The method ofclaim 47 wherein the inhibited SM38 activity is NAD+glycohydrolaseactivity.
 49. A method of treating a host infected with Schistosomacomprising administration of a SM38 inhibitor.
 50. The method of claim49 wherein the inhibited SM38 activity is NAD⁺ glycohydrolase activity.51. A method for stimulating a Schistosoma immunoprotective response ina host comprising immunizing said host with an SM38 polypeptide, orfragment thereof.
 52. A method for stimulating a Schistosomaimmunoprotective response in a host comprising immunizing said host witha nucleic acid molecule capable of encoding for a SM38 polypeptide, orfragment thereof.